Polypeptides and immunizing compositions containing gram positive polypeptides and methods of use

ABSTRACT

The present invention provides isolated polypeptides isolatable from a  Staphylococcus  spp. Also provided by the present invention are compositions that include one or more of the polypeptides, and methods for making and methods for using the polypeptides.

CONTINUING APPLICATION DATA

This application claims the benefit of U.S. Provisional Application Ser.No. 60/652,843, filed Feb. 14, 2005, which is incorporated by referenceherein.

BACKGROUND

Gram-positive bacteria are a remarkably diverse group of organisms thatcause a variety of diseases in both humans and animals. Some of thepathogens recognized as important in human and/or animal health includebacteria belonging to the families of Corynebacteriaceae,Enterococcacae, Micrococcaceae, Mycobacteriaceae, Nocardiaceae, andPeptococcaceae, which include such bacterial species as Actinomycesspp., Bifidobacterium spp., Corynebacterium spp., Enterococcus spp.,Erysipelothrix spp., Eubacterium spp., Kytococcus spp., Lactobacillusspp., Micrococcus spp., Mobiluncus spp., Mycobacteria spp.,Peptostreptococcus spp., Propionibacterium spp., and Staphylococcus spp.These pathogens cause a multitude of clinical manifestations in manydifferent animal species. The treatment for such infections hashistorically been antibiotics that attack the common structures andfunctions of gram-positive organisms. However, many of the moreubiquitous gram-positive organisms have developed resistance to severalclasses of antibiotics, making treatment of infections difficult. Thewidespread use of antibiotics in the treatment of bacterial diseases inboth humans and food production animals is likely a major contributingfactor in the proliferation of antibiotic-resistant strains of manyspecies of gram-positive organisms. Therefore, there is a great need tofind different treatments that prevent or eliminate infections bygram-positive organisms in animals as well as humans.

Staphylococcal Infections in Agricultural Animals

In the agricultural industry a number of important diseases are causedby gram-positive organisms. Examples of clinical conditions caused bygram positive bacterial infections include, mastitis, septicemia,pneumonia, osteomyelitis, meningoencephalitis, lymphangitis, dermatitis,genital tract infections, metritis, perinatal disease, pituitaryabscesses, arthritis, bursitis, orchitis, cystitis and pyelonephritis,caseous lymphadenitis, tuberculosis, ulcerative lymphangitis,erysipelas, laminitis, tyzzer's disease, tetanus, botulism, enteritis,malignant edema, braxy, bacillary hemoglobinuria, enterotoxemia.Staphylococcus spp., in particular, are capable of infecting manydifferent species of agricultural animals and can cause enormouseconomic losses. For example, the United States dairy industry isestimated to lose approximately $185 per cow annually due to mastitis, adisease often caused by Staphylococcus aureus. Since there are 9.5million head of milking cows in the U.S., the annual cost of mastitis isapproximately $1.8 billion. This is approximately 10% of the total valueof farm milk sales, and about two-thirds of this loss is due to reducedmilk production in sub-clinically infected cows. Other losses are due todiscarded abnormal milk and milk withheld from cows treated withantibiotic, costs of early replacement of affected cows, reduced salevalue of culled cows, costs of drugs and veterinary services, andincreased labor costs. In addition to its prevalence within the bovinedairy industry, mastitis caused by gram-positive cocci is also commonamong goats and sheep. Additional animal diseases caused by S. aureusinclude botryomycosis in horses, purulent synovitis and osteomyelitis inpoultry, snuffles in rabbits, abortions in swine, and tick pyemia inlambs. Other species of staphylococci are major skin pathogens of canine(S. intermedius) and swine (S. hycius). In poultry species,staphylococcal pathogens cause endorcarditis and septicemia.

Staphylococcal Infections in Humans

Staphylococcus spp. are also human pathogens causing a wide variety ofinfections. The species Staphylococcus aureus, a common colonizer ofhuman mucosa and skin, is an opportunistic pathogen that can causediverse human infections. For example, S. aureus is the causative agentof several skin infections, including impetigo, furunculosis,cellulitus, and scalded skin syndrome, as well as potentially fatalpost-surgical wound infections. In addition, the exposure ofimmunocompromised individuals to S. aureus in hospital settings hasresulted in organ infections such as pneumonia, urinary tractinfections, osteomyelitis, arthritis, bacteremia, and endocarditis. S.aureus is also the causative agent of toxinoses, most notably toxicshock syndrome and food poisoning. Food poisoning caused by thestaphylococcal enterotoxin B is the most common cause of food-borneillness, surpassing even salmonellosis, campylobacteriosis andlisteriosis. Other species of staphylococci also cause human disease; S.epidermidis, S. haemolyticus and S. hominis commonly infect implantedmedical devices and S. saprophyticus is associated with urinary tractinfections in women.

Virulence Mechanisms of Staphylococci

Staphylococci infect a variety of host tissues and evade the immunesystem through the production of several types of secreted proteins,surface expressed virulence factors and metabolic systems designed forsurvival amidst the limited resources and active defenses associatedwith the host environment. Colonization is the necessary first step inestablishing infection; numerous factors including capsule, lipoteichoicacid, and teichoic acid are common structural components contributing tocolonization. In addition, surface proteins such as staphylococcalfibronectin-binding protein and bone-sialoprotein binding proteinsspecifically bind host tissue components. Toxins are commonly producedamong staphylococcal pathogens and are highly damaging; several humandiseases, including food poisoning, toxic shock syndrome and exfoliativeskin conditions, are the direct result of extracellular secreted toxinproteins. A single isolate may encode genes for 20-30 different secretedtoxins. Some of the secreted protein products are superantigens that canbind nonspecifically to the MHC class II molecule of anantigen-presenting cell and, simultaneously, to the T-cell receptor of aT cell. The binding induces T cell signaling and leads to the release ofhigh levels of proinflammatory factors, ultimately inducing host damagedue to the overwhelming immune response. Another class of virulencefactors expressed on the surface disguise the bacteria from the hostimmune system. For example, the S. aureus surface-expressed Protein Ainhibits opsonization and phagocytosis by binding of the Fc component ofhost antibody. Numerous proteases, hemolysins (alpha, beta, gamma anddelta), nucleases, lipases, hyaluronidase, and collagenase also aidbacteria in extracting nutrients from surrounding cells and protectingthem against host defenses.

Antibiotic Resistance Among Staphylococci

The CDC estimates that each year nearly 2 million people in the UnitedStates acquire a nosocomial infection, resulting in 90,000 deathsannually. Of these fatal infections, 70% are caused byantibiotic-resistant bacteria. The increase in antibiotic-resistanceamong microbial species is particularly pronounced in skin and mucosalcolonizers such as S. aureus. For example, the vast majority of S.aureus isolated from hospital settings are resistant to penicillin, and50% are also resistant to the semisynthetic penicillins, such asmethicillin, nafcillin, and oxacillin. These isolates, referred to asMRSA (methicillin resistant S. aureus) were first seen in the 1970s, andare now firmly established in hospital settings. Recently there havebeen several cases of MRSA infections in the community, where theinfected individuals had no previous exposure to hospitals or healthcareworkers. This alarming trend is intensified by the isolation of MRSAisolates that are less susceptible to vancomycin, a glycopeptide used totreat MRSA. Very few strains have been shown to be truly resistant tovancomycin according to the CDC's definition of vancomycin resistance,but several MRSA strains have been characterized as consisting ofsubpopulations with reduced susceptibility to vancomycin, or VISA(vancomycin intermediate S. aureus). Since the isolation of vancomycinresistant and vancomycin intermediate strains is a relatively newdevelopment, there is little data concerning their prevalence inhospitals and/or the community. Occasionally, VRSA (vancomycin resistantS. aureus) with full resistance to vancomycin and carrying a resistanceplasmid likely acquired from Enterococcus spp. have also been recoveredfrom humans.

Strategies for the Prevention and Treatment of Staphylococcus Infections

The emergence of numerous gram-positive pathogens that are resistant tomultiple antibiotics has fueled research efforts aimed at developingpreventative vaccines to protect against disease. Vaccines are designedto be administered to patients in order to elicit a long-term memoryresponse from the immune system, so that if the pathogen is encounteredat a future time, the immune system can more quickly and efficientlyclear the pathogen. To date, a broadly-protective vaccine againstgram-positive pathogens associated with a number of severe humandiseases, particularly those disease associated with staphylococcalinfections, is not available. Vaccine development approaches for theprevention of staphylococcal infections include those reporting the useof microbial surface components recognizing adhesion matrix molecules[MSCRAMMS (Nilsson et al. 1998. J Clin Invest 101:2640-9; Menzies et al.2002. J Infect Dis 185:937-43; Fattom et al. 2004. Vaccine 22:880-7],surface polysaccharides (McKenney et al. 2000; McKenney et al. 1999.Science 284:1523-7; Maira-Litran et al. 2002. Infect Immun 70:4433-40;Maira-Litran et al. 2004. Vaccine 22:872-9; Maira-Litran et al. 2005.Infect Immun 73:6752-62) and mutated exoproteins (Lowell et al. 1996.Infect Immun 64:4686-93; Stiles et al. 2001. Infect Immun 69:2031-6;Gampfer et al. 2002. Vaccine 20:3675-84), as antigens in subunit vaccinecompositions, as well as one live avirulent strain (Reinoso et al. 2002.Can J Vet Res 66:285-8) and several DNA vaccine approaches (Ohwada etal. 1999. J Antimicrob Chemother 44:767-74); Brouillette et al. 2002.Vaccine 20:2348-57; Senna et al. 2003. Vaccine 21:2661-6). Although manyof these compositions have shown some degree of protection, they haveachieved little cross-protection against diverse staphylococcal strainsand have additionally failed to elicit substantial immune responses inimmunocompromised patients, an important at-risk population fornosocomial infections.

The most severe staphylococcal diseases are those mediated by theaforementioned supernantigenic pyrogenic exotoxins (SPEs) thatnonspecifically stimulate T-cells independent of antigen presentation.Such diseases include toxic shock syndrome, exfoliative skin disease,and possibly Kawasaki syndrome. For these SPE-mediated diseases,immunotherapeutic agents that boost the immune system during an activeinfection are often more effective than vaccines, which are typicallyadministered prior to infection. The overwhelming nature of the immuneresponse to SPE necessitates rapid reduction in toxin activity as thefirst objective in therapy. To date, toxin neutralization in S.aureus-mediated disease has been most effectively accomplished by theadministration of intravenous human immunoglobulin (IVIG), a purified,concentrated human antibody preparation from several thousand humandonors (Takei et al. 1993. J Clin Invest 91:602-7; Stohl and Elliot.1996. Clin Immunol Immunopathol 79:122-33). The widespread distributionof S. aureus, which colonizes approximately 30% of healthy human adults,coincides with high exposure rates for the majority of the population,so the level of anti-staphylococcal anti-toxin antibodies in IVIG isoften sufficient to neutralize toxin long enough to stabilize the immuneresponse until the bacterial load is reduced with antibiotics(Schlievert, 2001. J Allergy Clin Immunol 108(4 Suppl):S107-110). IVIGpreparations from multiple manufacturers have been shown to neutralizetoxin in proliferation assays with human peripheral blood mononuclearcells, inhibit toxin-induced human T cell-driven B cell differentiationin vitro (Stohl and Elliot. 1996. Clin Immunol Immunopathol 79:122-33;Stohl and Elliott. 1995. J Immunol 155:1838-50; Stohl et al. 1994. JImmunol 153:117-27) and reduce IL-4 and IL-2 secretion in PBMCsstimulated with staphylococcal enterotoxin B (Takei et al. 1993. J ClinInvest 91:602-7; Darenberg et al. 2004. Clin Infect Dis 38:836-42). IVIGtherapy, with its proven ability to neutralize SPE, is now a recommendedtherapy for Kawasaki syndrome and is gaining favor as a treatment methodfor staphylococcal toxic shock syndrome (Schlievert 2001. J Allergy ClinImmunol 108(4 Suppl):S107-110). Use of IVIG as an immunoprotective woundlavage during surgery has also been investigated in mice (Poelstra etal. 2000. Tissue Eng 6(4):401-411). Although standard IVIG has utilityfor limiting the advance of some staphylococcal SPE-mediated disease,the safety, efficacy and consistency of human IVIG preparationsgenerated from thousands of unselected human donors remainscontroversial (Baker et al. 1992. N EngI J Med 327:213-9; Miller et al.2001. J Allergy Clin Immunol 108:S91-4; Sacher, 2001. J Allergy ClinImmunol 108:S139-46; Darenberg et al. 2004. Clin Infect Dis 38:836-42).Furthermore, the benefit of IVIG in preventing some staphylococcalinfections is doubtful (Baker et al. 1992. N EngI J Med 327:213-9; Hill,H. R. 2000. J Pediatr 137:595-7; Darenberg et al. 2004. Clin Infect Dis38:836-42). In order to increase the effectiveness of IVIG in treatingstaphylococcal infections in certain at-risk populations, aplasma-derived, donor-selected, polyclonal anti-staphylococcal human IgGwith high titers of antibody directed toward the staphylococcal MSCRAMMSclumping factor A (CIfA) and fibrinogen-binding protein G (SdrG) wascreated and tested with success in very low birthweight infants toprevent staphylococcal sepsis (Vernachio et al. 2003. Antimicrob AgentsChemother 47:3400-6; Bloom et al. 2005. Pediatr Infect Dis J 24:858-866;Capparelli et al. 2005. Antimicrob Agents Chemother 49:4121-7). Aspecific humanized monoclonal antibody toward the S. aureus MSCRAMMClumping factor A, is also being developed. The antibody was selectedfrom a pool of thousands of murine anti-ClfA antibodies for its abilityto bind ClfA in a manner that abrogates S. aureus binding to humanfibronectin and was subsequently humanized by mutating specific targetedresidues to mimic the homologous human germline subgroup antibody (Hallet al. 2003. Infect Immun 71:6864-70; Domanski et al. 2005. Infect Immun73:5229-32). The specific antibody is being designed for use inconjunction with antibiotics for the treatment of severelife-threatening S. aureus infection, although animal studies alsodemonstrated a prophylactic protective effect.

SUMMARY

The present invention provides compositions including two or moreisolated polypeptides. The two isolated polypeptides may have amolecular weight of 88 kDa, 55 kDa, 38 kDa, 37 kDa, 36 kDa, 35 kDa, 33kDa, or a combination thereof. For instance, a composition may includeisolated proteins of 88 kDa and 55 kDa. In some aspects the compositionmay include isolated polypeptides having molecular weights of 88 kDa, 55kDa, 38 kDa, 37 kDa, 36 kDa, 35 kDa, and 33 kDa. The molecular weight isdetermined by electrophoresis on a sodium dodecyl sulfate-polyacrylamidegel. The polypeptides are isolatable from a Staphylococcus aureus whenincubated in media including an iron chelator and not isolatable whengrown in the media without the iron chelator. The composition protectsan animal, such as a mouse or cow or human, against challenge with an S.aureus strain, for instance ATCC strain 19636. The composition mayfurther include a pharmaceutically acceptable carrier, and may furtherinclude an isolated polypeptide having a molecular weight of 150 kDa,132 kDa, 120 kDa, 75 kDa, 58 kDa, 50 kDa, 44 kDa, 43 kDa, 41 kDa, 40kDa, or a combination thereof, and isolatable from a S. aureus whengrown in the media without the iron chelator. In some aspects thepolypeptides of the composition may be isolated from S. aureus ATCCstrain 19636.

The present invention also provides methods for using the compositions.In one aspect the method is for treating in infection in a subject, andincludes administering an effective amount of a composition of thepresent invention to a subject having or at risk of having an infectioncaused by a Staphylococcus spp. In another aspect, the method is fortreating a symptom in a subject, and it includes administering aneffective amount of a composition of the present invention to a subjecthaving an infection caused by a Staphylococcus spp. The subject may be amammal, such as a human, horse, or cow. The Staphylococcus spp. may beS. aureus.

The present invention further provides methods for using antibody, forinstance, polyclonal antibody, that specifically binds polypeptides ofthe present invention. In one aspect, the method is for treating aninfection in a subject, and includes administering an effective amountof a composition to a subject having or at risk of having an infectioncaused by a Staphylococcus spp., wherein the composition includesantibody that specifically binds two isolated polypeptides of thepresent invention. In another aspect, the method is for treating asymptom in a subject, and includes administering an effective amount ofa composition to a subject having an infection caused by aStaphylococcus spp., wherein the composition includes antibody thatspecifically binds two isolated polypeptides of the present invention.The subject may be a mammal, such as a human, horse, or cow. TheStaphylococcus spp. may be S. aureus.

Also provided by the present invention are methods for decreasingcolonization in a subject. In one aspect, the method includesadministering an effective amount of a composition of the presentinvention to a subject colonized by a Staphylococcus spp. In anotheraspect, the method includes administering an effective amount of acomposition to a subject colonized by Staphylococcus spp., wherein thecomposition includes antibody that specifically binds two isolatedpolypeptides of the present invention.

The present invention provides a kit for detecting antibody thatspecifically binds a polypeptide. The kit includes, in separatecontainers, an isolated polypeptide of the present invention, and areagent that detects an antibody that specifically binds thepolypeptide.

The present invention further provides a composition including twoisolated polypeptides having molecular weights selected from 88 kDa, 55kDa, 38 kDa, 37 kDa, 36 kDa, 35 kDa, and 33 kDa, wherein molecularweight is determined by electrophoresis on a sodium dodecylsulfate-polyacrylamide gel. Each polypeptide of the composition has amass fingerprint of at least 80% similarity to a mass fingerprint of apolypeptide of the same molecular weight polypeptide expressed byStaphylococcus aureus ATCC strain 19636, wherein the polypeptide isisolatable from a Staphylococcus aureus when incubated in mediacomprising an iron chelator and not isolatable when grown in the mediawithout the iron chelator. For instance, the isolated polypeptide with amolecular weight of 88 kDa has a mass fingerprint of at least 80%similarity to a mass fingerprint of a 88 kDa polypeptide expressed byStaphylococcus aureus ATCC strain 19636, and the isolated polypeptidewith a molecular weight of 55 kDa has a mass fingerprint of at least 80%similarity to a mass fingerprint of a 55 kDa polypeptide expressed byStaphylococcus aureus ATCC strain 19636.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The electrophoretic profile of the proteins of different strainsStaphylococcus aureus derived from different species grown with andwithout iron (lanes marked Fe++ and DP, respectively).

FIG. 2. The difference in mortality between vaccinated andnon-vaccinated mice after homologous and heterologous challenge withStaphylococcus aureus.

FIG. 3. Kaplan-Meier survival curve showing percent survival aftervaccination and homologous challenge with S. aureus ATCC 19636.

FIG. 4. Kaplan-Meier survival curve showing percent survival aftervaccination and heterologous challenge with S. aureus ATCC 19636.

FIG. 5. The Kaplan-Meier survival curve showing percent survival afterpassive immunization and homologous challenge with S. aureus ATCC 19636.

FIG. 6. The Kaplan-Meier survival curve showing percent survival afterpassive immunization and heterologous challenge with S. aureus strain1477.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides polypeptides and compositions includingpolypeptides. As used herein, “polypeptide” refers to a polymer of aminoacids linked by peptide bonds. Thus, for example, the terms peptide,oligopeptide, protein, and enzyme are included within the definition ofpolypeptide. This term also includes post-expression modifications ofthe polypeptide, such as glycosylations, acetylations, phosphorylations,and the like. The term polypeptide does not connote a specific length ofa polymer of amino acids. A polypeptide may be isolatable directly froma natural source, or can be prepared with the aid of recombinant,enzymatic, or chemical techniques. In the case of a polypeptide that isnaturally occurring, such a polypeptide is typically isolated. An“isolated” polypeptide is one that has been removed from its naturalenvironment. For instance, an isolated polypeptide is a polypeptide thathas been removed from the cytoplasm or from the membrane of a cell, andmany of the polypeptides, nucleic acids, and other cellular material ofits natural environment are no longer present. An “isolatable”polypeptide is a polypeptide that could be isolated from a particularsource. A “purified” polypeptide is one that is at least 60% free,preferably at least 75% free, and most preferably at least 90% free fromother components with which they are naturally associated. Polypeptidesthat are produced outside the organism in which they naturally occur,e.g., through chemical or recombinant means, are considered to beisolated and purified by definition, since they were never present in anatural environment. As used herein, a “polypeptide fragment” refers toa portion of a polypeptide that results from digestion of a polypeptidewith a protease. Unless otherwise specified, “a,” “an,” “the,” and “atleast one” are used interchangeably and mean one or more than one. Theterms “comprises” and variations thereof do not have a limiting meaningwhere these terms appear in the description and claims.

A polypeptide of the present invention may be characterized by molecularweight, mass fingerprint, or the combination thereof. The molecularweight of a polypeptide, typically expressed in kilodaltons (kDa), canbe determined using routine methods including, for instance, gelfiltration, gel electrophoresis including sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE), capillary electrophoresis,mass spectrometry, and liquid chromatography including HPLC. Preferably,molecular weight is determined by resolving a polypeptide using an SDSpolyacrylamide gel having a stacking gel of about 4% and a resolving gelof about 10% under reducing and denaturing conditions. Unless indicatedotherwise, molecular weight refers to molecular weight as determined bySDS-PAGE. As used herein, a “mass fingerprint” refers to a population ofpolypeptide fragments obtained from a polypeptide after digestion with aprotease. Typically, the polypeptide fragments resulting from adigestion are analyzed using a mass spectrometric method. Eachpolypeptide fragment is characterized by a mass, or by a mass (m) tocharge (z) ratio, which is referred to as an “m/z ratio” or an “m/zvalue”. Methods for generating a mass fingerprint of a polypeptide areroutine. An example of such a method is disclosed in Example 13.

Polypeptides of the present invention may be metal regulatedpolypeptides. As used herein, a “metal regulated polypeptide” is apolypeptide that is expressed by a microbe at a greater level when themicrobe is grown in low metal conditions compared to growth of the samemicrobe in high metal conditions. Low metal and high metal conditionsare described herein. For instance, one class of metal regulatedpolypeptide produced by Staphylococcus spp. is not expressed atdetectable levels during growth of the microbe in high metal conditionsbut is expressed at detectable levels during growth in low metalconditions. Examples of such metal regulated polypeptides isolatablefrom S. aureus after growth in low iron conditions have molecularweights of 88 kDa, 55 kDa, 38 kDa, 37 kDa, 36 kDa, 35 kDa, and 33 kDa.Examples of such metal regulated polypeptides isolatable from S. aureusafter growth in low zinc or low copper conditions have molecular weightsof 115 kDa, 88 kDa, 80 kDa, 71 kDa, 69 kDa, 35 kDa, 30 kDa, 29, kDa, and27 kDa.

The present invention also includes polypeptides that are not metalregulated. Such polypeptides are expressed in the presence of a metalion such as ferric chloride, and also expressed when grown in low ironconditions. Examples of such polypeptides isolatable from S. aureus havemolecular weights of 150 kDa, 132 kDa, 120 kDa, 75 kDa, 58 kDa, 50 kDa,44 kDa, 43 kDa, 41 kDa, and 40 kDa.

Whether a polypeptide is a metal regulated polypeptide or not can bedetermined by methods useful for comparing the presence of polypeptides,including, for example, gel filtration, gel electrophoresis includingsodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),capillary electrophoresis, mass spectrometry, and liquid chromatographyincluding HPLC. Separate cultures of a microbe are grown under highmetal conditions and under low metal conditions, polypeptides of thepresent invention are isolated as described herein, and the polypeptidespresent in each culture are resolved and compared. Typically, an equalamount of polypeptides from each culture is used. Preferably, thepolypeptides are resolved using an SDS polyacrylamide gel having astacking gel of about 4% and a resolving gel of about 10% under reducingand denaturing conditions. For instance, 30 micrograms (μg) of totalpolypeptide from each culture may be used and loaded into wells of agel. After running the gel and staining the polypeptides with CoomasieBrilliant Blue, the two lanes can be compared. When determining whethera polypeptide is or is not expressed at a detectable level, 30 μg oftotal polypeptide from a culture is resolved on an SDS-PAGE gel andstained with Coomasie Brilliant Blue using methods known in the art. Apolypeptide that can be visualized by eye is considered to be expressedat a detectable level, while a polypeptide that cannot be visualized byeye is considered to not be expressed at a detectable level.

Polypeptides of the present invention may have immunogenic activity.“Immunogenic activity” refers to the ability of a polypeptide to elicitan immunological response in an animal. An immunological response to apolypeptide is the development in an animal of a cellular and/orantibody-mediated immune response to the polypeptide. Usually, animmunological response includes but is not limited to one or more of thefollowing effects: the production of antibodies, B cells, helper Tcells, suppressor T cells, and/or cytotoxic T cells, directed to anepitope or epitopes of the polypeptide. “Epitope” refers to the site onan antigen to which specific B cells and/or T cells respond so thatantibody is produced. The immunogenic activity may be protective.“Protective immunogenic activity” refers to the ability of a polypeptideto elicit an immunological response in an animal that prevents orinhibits infection by Staphylococcus spp., for instance, S. aureus.Whether a polypeptide has protective immunogenic activity can bedetermined by methods known in the art, for instance as described inExamples 5, 9, or 12. For example, a polypeptide of the presentinvention, or combination of polypeptides of the present invention,protect a rodent such as a mouse against challenge with a Staphylococcusspp. A polypeptide of the present invention may have seroactiveactivity. “Seroactive activity” refers to the ability of a candidatepolypeptide to react with antibody present in convalescent serum from ananimal infected with a Staphylococcus spp., for instance, S. aureus. Insome aspects, the convalescent serum may be from an animal infected withthe ATCC isolate 19636, strain SAAV1, strain 2176, or strain 1477.Polypeptides of the present invention may have immunoregulatoryactivity. “Immunoregulatory activity” refers to the ability of apolypeptide to act in a nonspecific manner to enhance an immune responseto a particular antigen. Methods for determining whether a polypeptidehas immunoregulatory activity are known in the art.

A polypeptide of the present invention may have the characteristics of apolypeptide expressed by a reference microbe. The characteristics caninclude both molecular weight and mass fingerprint. The referencemicrobe can be a gram positive, preferably a member of the familyMicrococcaceae, preferably, Staphylococcus spp., more preferably,Staphylococcus aureus. Preferred examples of strain are detailed inTable 1.

TABLE 1 Bacterial Strains. Bacterial cell Laboratory designation S.aureus ATCC isolate 19636 S. aureus strain SAAV1 S. aureus strain 1477S. aureus strain 2176

When the reference microbe is S. aureus ATCC isolate 19636, a candidatepolypeptide is considered to be a polypeptide of the present inventionif it has a molecular weight of 88 kDa, 55 kDa, 38 kDa, 37 kDa, 36 kDa35 kDa, or 33 kDa, and has a mass fingerprint that is similar to themass fingerprint of a metal regulated polypeptide expressed by areference microbe and having a molecular weight of 88 kDa, 55 kDa, 38kDa, 37 kDa, 36 kDa 35 kDa, or 33 kDa, respectively. Preferably, suchpolypeptides are metal regulated. For instance, a candidate polypeptideis a polypeptide of the present invention if it has a molecular weightof 88 kDa and has a mass fingerprint similar to the mass fingerprint ofan 88 kDa metal regulated polypeptide produced by the reference strainS. aureus ATCC isolate 19636.

When the reference microbe is S. aureus isolate SAAV1, a candidatepolypeptide is considered to be a polypeptide of the present inventionif it has a molecular weight (as determined by SDS-PAGE) of 88 kDa, 55kDa, 38 kDa, 37 kDa, 36 kDa, 35 kDa, or 33 kDa, and has a massfingerprint that is similar to the mass fingerprint of a polypeptideexpressed by a reference microbe and having a molecular weight (asdetermined by SDS-PAGE) of 88 kDa, 55 kDa, 38 kDa, 37 kDa, 36 kDa, 35kDa, or 33 kDa, respectively. Preferably, such polypeptides are metalregulated. For instance, a candidate polypeptide is a polypeptide of thepresent invention if it has a molecular weight of 88 kDa and has a massfingerprint similar to the mass fingerprint of an 88 kDa metal regulatedpolypeptide produced by the reference strain S. aureus isolate SAAV1.

When the reference microbe is S. aureus strain 2176, a candidatepolypeptide is considered to be a polypeptide of the present inventionif it has a molecular weight (as determined by SDS-PAGE) of 88 kDa, 80kDa, 65 kDa, 55 kDa, 37 kDa, 36 kDa, 35 kDa, 33 kDa, or 32 kDa, and hasa mass fingerprint that is similar to the mass fingerprint of apolypeptide expressed by a reference microbe and having a molecularweight (as determined by SDS-PAGE) of 88 kDa, 80 kDa, 65 kDa, 55 kDa, 37kDa, 36 kDa, 35 kDa, 33 kDa, or 32 kDa, respectively. Preferably, suchpolypeptides are metal regulated. For instance, a candidate polypeptideis a polypeptide of the present invention if it has a molecular weightof 88 kDa and has a mass fingerprint similar to the mass fingerprint ofan 88 kDa metal regulated polypeptide produced by the reference strainS. aureus isolate 2176.

When the reference microbe is S. aureus strain 1477, a candidatepolypeptide is considered to be a polypeptide of the present inventionif it has a molecular weight (as determined by SDS-PAGE) of 88 kDa, 80kDa, 65 kDa, 55 kDa, 37 kDa, 36 kDa, 35 kDa, 33 kDa, or 32 kDa, and hasa mass fingerprint that is similar to the mass fingerprint of apolypeptide expressed by a reference microbe and having a molecularweight (as determined by SDS-PAGE) of 88 kDa, 80 kDa, 65 kDa, 55 kDa, 37kDa, 36 kDa, 35 kDa, 33 kDa, or 32 kDa, respectively. Preferably, suchpolypeptides are metal regulated. For instance, a candidate polypeptideis a polypeptide of the present invention if it has a molecular weightof 88 kDa and has a mass fingerprint similar to the mass fingerprint ofan 88 kDa metal regulated polypeptide produced by the reference strainS. aureus isolate 1477.

The polypeptides expressed by a reference microbe and referred to aboveby molecular weight can be obtained by growth of the reference microbeunder low metal conditions and the subsequent isolation of a polypeptideby the processes disclosed herein. A candidate polypeptide is isolatablefrom a microbe, preferably a gram positive microbe, more preferably, amember of the family Micrococcaceae, preferably, Staphylococcus spp.,more preferably, Staphylococcus aureus.

Other gram positive microbes from which polypeptides can be isolatedinclude Corynebacterium spp., Enterococcus spp., Erysipelothrix spp.,Kytococcus spp., and Micrococcus spp., Mycobacterium spp., andErysipelothrix spp. A candidate polypeptide may also be produced usingrecombinant, enzymatic, or chemical techniques.

A candidate polypeptide may be evaluated by mass spectrometric analysisto determine whether the candidate polypeptide has a mass fingerprintsimilar to one of the polypeptides expressed by a reference microbe andreferred to above by molecular weight. Typically, the candidatepolypeptide is isolated, for instance by resolving the candidatepolypeptide by gel electrophoresis and excising the portion of the gelcontaining the candidate polypeptide. Any gel electrophoresis methodthat separates polypeptides based on differing characteristics can beused, including 1 dimensional or 2 dimensional gel electrophoresis, aswell as liquid chromatographic separation based on, for instance,hydrophobicity, pI, or size. The candidate polypeptide is fragmented,for instance by digestion with a protease. Preferably, the proteasecleaves the peptide bond on the carboxy-terminal side of the amino acidlysine and the amino acid arginine, except when the amino acid followingthe lysine or the arginine is a proline. An example of such a proteaseis trypsin. Methods for digesting a polypeptide with trypsin are routineand known in the art. An example of such a method is disclosed inExample 13.

Methods for the mass spectrometric analysis of polypeptides are routineand known in the art and include, but are not limited to, matrixassisted laser desorption/ionization time of flight mass spectroscopy(MALDI-TOF MS). Typically, a mixture containing the polypeptidefragments obtained from a candidate polypeptide is mixed with a matrixthat functions to transform the laser energy to the sample and produceionized, preferably monoisotopic, polypeptide fragments. Examples ofmatrices that can be used include, for instance, sinapinic acid orcyano-4-hydroxycinnamic acid. An example of a method for the analysis ofpolypeptides by MALDI-TOF MS is described in Example 13. The ionizedpolypeptide fragments are separated according to their m/z ratio, anddetected to yield a spectrum of m/z ratio versus intensity. The spectrumincludes m/z values that represent the polypeptide fragments derivedfrom the candidate polypeptide. For any given polypeptide, the amount ofeach polypeptide fragment resulting from a trypsin digestion should beequimolar. However, it is known that trypsin digestion is not always100% efficient, for instance, some sites are more efficiently cleaved.Thus, when MALDI-TOF MS is used to determine m/z values, the intensityof each m/z value is typically not identical. Generally, a spectrum hasa background level of noise present across most of the x-axis (i.e., theaxis having the values of the m/z ratios). This background level ofnoise varies depending on the running conditions and the machine used,and is easily identified by visual inspection of the spectrum. An m/zvalue is generally considered to represent a polypeptide fragment whenthe intensity is at least 2 times greater, at least 3 times greater, orat least 4 times greater than the background level of noise. Thespectrum usually includes other m/z values that are artifacts resultingfrom, for instance, incomplete digestion, over digestion, otherpolypeptides that may be present in the mixture, or the protease used todigest the polypeptide including m/z values resulting from autolysis ofthe protease. This method of digesting a polypeptide with a protease isrecognized in the art as resulting in a mass fingerprint of greatspecificity that can be used to accurately characterize the polypeptideand distinguish it from other polypeptides.

In this aspect of the invention, when a candidate polypeptide isanalyzed by mass spectroscopy, preferably both the candidate polypeptideand the polypeptide from the reference microbe are prepared and analyzedtogether, thereby decreasing any potential artifacts resulting fromdifferences in sample handling and running conditions. Preferably, allreagents used to prepare and analyze the two polypeptides are the same.For instance, the polypeptide from the reference microbe and thecandidate polypeptide are isolated under substantially the sameconditions, fragmented under substantially the same conditions, andanalyzed by MALDI-TOF MS on the same machine under substantially thesame conditions. A mass fingerprint of a candidate polypeptide isconsidered to be similar to the mass fingerprint of a polypeptide from areference microbe when at least 80%, at least 90%, at least 95%, orsubstantially all of the m/z values present in the spectrum of thereference microbe polypeptide and above the background level of noiseare also present in the spectrum of the candidate polypeptide.

In another aspect, a polypeptide is considered to be a polypeptide ofthe present invention if it has a molecular weight of a referencepolypeptide described in Table 2, 3, 4, or 5 and has a mass fingerprintthat includes the population of polypeptide fragments of the referencepolypeptide as listed in Table 2, 3, 4, or 5. For instance, apolypeptide of the present invention includes a polypeptide of 88 kDaand a mass fingerprint that includes polypeptide fragments having massesof HVDVR, YSYER, IIGDYRR, IFTDYRK, ELKELGQK, YAQVKPIR, QMQFFGAR,SMQPFGGIR, VSGYAVNFIK, NHATAWQGFK, LWEQVMQLSK, SLGKEPEDQNR,DGISNTFSIVPK, AGVITGLPDAYGR, TSTFLDIYAER, SMQPFGGIRMAK, THNQGVFDAYSR,KAGVITGLPDAYGR, TLLYAINGGKDEK, IEMALHDTEIVR, AGEPFAPGANPMHGR,VALYGVDFLMEEK, KTHNQGVFDAYSR, YGFDLSRPAENFK, TSSIQYENDDIMR,KAGEPFAPGANPMHGR, RVALYGVDFLMEEK, LWEQVMQLSKEER, MLETNKNHATAWQGFK,MHDFNTMSTEMSEDVIR, YGNNDDRVDDIAVDLVER, ETLIDAMEHPEEYPQLTIR,YAQVKPIRNEEGLVVDFEIEGDFPK. The mass fingerprint of a candidatepolypeptide can be determined by a mass spectrometric method, forinstance by MALDI-TOF MS. The mass fingerprint of a candidatepolypeptide will generally have additional polypeptide fragments andtherefore additional m/z values other than those listed for apolypeptide in Table 2, 3, 4, or 5. Preferably, when the candidatepolypeptide is being compared to a polypeptide in Table 2, 3, 4, or 5,the candidate polypeptide is isolatable from a microbe, preferably agram positive microbe, more preferably, a member of the familyMicrococcaceae, preferably, Staphylococcus spp., more preferably,Staphylococcus aureus. Other gram positive microbes includeCorynebacterium spp., Enterococcus spp., Erysipelothrix spp., Kytococcusspp., Listeria spp., Micrococcus spp., and Mycobacterium spp., andErysipelothrix spp. A candidate polypeptide can be obtained by growth ofa microbe under low metal conditions and the subsequent isolation of apolypeptide by the processes described herein.

It is well known in the art that modifications of amino acids can beaccidentally introduced during sample handling, such as oxidation, andformation of carbamidomethyl derivatives. Further, these types ofmodifications alter the m/z value of a polypeptide fragment. Forinstance, if a polypeptide fragment contains a methionine that isoxidized, the m/z value will be increased by 16 relative to the samefragment that does not contain the oxidized methionine. Accordingly,those polypeptide fragments in Tables 2, 3, 4, or 5 having the notation“oxidation (M)” have an m/z value that is increased by 16 relative tothe same fragment that does not contain the oxidized methionine. It isunderstood that the polypeptide fragments of Table 2, 3, 4, or 5 can bemodified during sample handling.

TABLE 2 Characteristics of polypeptides obtained from S. aureus ATCCisolate 19636. Approximate m/z value of polypeptide Polypeptidemolecular weight in fragments resulting from Predicted amino acidsequence designation kilodaltons (kDa)¹ trypsin digest² of thepolypeptide fragment P23 88 625.4 HVDVR 717.3 YSYER 892.5 IIGDYRR 942.5IFTDYRK 944.5 ELKELGQK 974.6 YAQVKPIR 984.5 QMQFFGAR 992.5 SMQPFGGIR1097.6 VSGYAVNFIK 1159.5 NHATAWQGFK 1261.7 LWEQVMQLSK 1272.7 SLGKEPEDQNR1277.7 DGISNTFSIVPK 1289.7 AGVITGLPDAYGR 1315.7 TSTFLDIYAER 1322.7SMQPFGGIRMAK 1394.7 THNQGVFDAYSR 1417.8 KAGVITGLPDAYGR 1421.8TLLYAINGGKDEK 1426.8 IEMALHDTEIVR 1508.8 AGEPFAPGANPMHGR 1513.9VALYGVDFLMEEK 1522.8 KTHNQGVFDAYSR 1543.9 YGFDLSRPAENFK 1571.8TSSIQYENDDIMR 1636.9 KAGEPFAPGANPMHGR 1670.0 RVALYGVDFLMEEK 1676.0LWEQVMQLSKEER 1876.2 MLETNKNHATAWQGFK 2043.1 MHDFNTMSTEMSEDVIR 2078.2YGNNDDRVDDIAVDLVER 2285.5 ETLIDAMEHPEEYPQLTIR 2892.9YAQVKPIRNEEGLVVDFEIEGDFPK P25 55 783.6 LHSWLK 911.7 KLHSWLK 937.6TYTFHLR 996.6 KFDGTGPFK 1025.6 QAIGHMVNR 1063.6 KWDVSEDGK 1185.6IYNSIDDAFK 1277.6 NLEMAMYYDK 1324.7 ENKQLTYTTVK 1346.7 AESLLDEAGWKK1381.8 TVRQAIGHMVNR 1394.8 TYTFHLRDDVK 1400.7 KGETNFAFTDDR 1419.7FHDGTPFDADAVK 1422.8 NVTDINFDMPTR 1428.8 DKIYNSIDDAFK 1483.8EQAEYLQAEFKK 1509.8 VMPAGETAFLSMKK 1547.9 FHDGTPFDADAVKK 1550.9NVTDINFDMPTRK 1559.9 LNINGETSDKIAER 1788.1 EILDGQEKPATQLFAK 1930.1GSSSQKEQAEYLQAEFK 1946.0 DESADFNKNDQYWGEK 2100.4 IAKEILDGQEKPATQLFAK2239.3 VSFTQSQYELPFNEMQYK 2493.5 EAYQPALAELAMPRPYVFVSPK + Oxidation (M)2900.6 DIGDMNPHVYGGSMSAESMIYEPLVR + 2 Oxidation (M) 2916.6DIGDMNPHVYGGSMSAESMIYEPLVR + 3 Oxidation (M) P26 38 993.6 IVYVGADEK996.7 QALNNPVLK 1237.7 ETVKIENNYK 1272.7 ENPDVILAMDR 1502.0IAATKPEVIFISGR 1507.9 NAVVLDYGALDVMK 1523.9 ALPNFLESFKDDK 1559.9LWYFAAGSTTTTIK 1716.0 FGGLVYDTLGFNAVDK 1737.0 IVYVGADEKNLIGSMK 1844.1FGGLVYDTLGFNAVDKK 1929.1 GRFGGLVYDTLGFNAVDK 1998.2 TVMYLLVNEGELSTFGPK2234.4 EVNFDKIAATKPEVIFISGR 3143.8 VSNSNHGQNVSNEYVNKENPDVILAMDR P27 37699.5 FEYIK 729.4 DAWPLK 792.5 ASVVNFR 852.4 VYDQLSK 987.5 HAMGTTEIK1008.5 LIDDLYEK 1020.5 YKDAWPLK 1074.5 EKEAEDLLK 1083.6 LKPDLIVASK1169.5 FEYIKNDLK 1182.5 KTESEWTSSK 1184.5 YDDKVAAFQK 1223.5 NEKVYDQLSK1278.6 IAPTVSTDTVFK 1497.6 TESEWTSSKEWK 1502.7 DAWPLKASVVNFR 1558.8QVDNGKDIIQLTSK 1605.8 LIDDLYEKLNIEK 1623.8 IVGQEPAPNLEEISK 1712.8ESIPLMNADHIFVVK 1800.9 IYAGGYAGEILNDLGFK 1957.0 IYAGGYAGEILNDLGFKR2252.0 NNQVSDDLDEITWNLAGGYK 3383.9 RVVTLYQGATDVAVSLGVKPVGAVESWTQKPK P2836 646.4 DVWAR 725.5 IIKPVR 1068.4 IGDYTSVGTR 1185.5 KQPNLEEISK 1327.6LKPDLIIADSSR 1343.6 VDIVDRDVWAR 2080.9 GPYLQLDTEHLADLNPER 2438.1AGLLAHPNYSYVGQFLNELGFK 2789.4 IVVLEYSFADALAALDVKPVGIADDGK P29 35 760.5AGWAEVK 1012.6 TVDIPKDPK 1107.6 KDWEETTAK 1204.7 VAPTVVVDYNK 1238.6YLEQQEMLGK 1244.6 LYTYGDNWGR 1259.7 IAVVAPTYAGGLK 1281.7 GGEVLYQAFGLK1516.8 AGWAEVKQEEIEK 1683.9 LGANIVAVNQQVDQSK 1877.1 EKPDLIIVYSTDKDIK1884.0 AIGQDATVSLFDEFDKK 2227.1 VDAGTYWYNDPYTLDFMR 2781.4YAGDYIVSTSEGKPTPGYESTNMWK P30 33 834.5 QAIEFVK 864.5 YIAQLEK 946.5QGTPEQMR 962.5 QAIEFVKK 976.5 DKFNDIPK 1054.5 AMITSEGAFK 1202.5SNIETVHGSMK 1268.6 HLLVETSVDKK 1443.6 DIFGEVYTDSIGK 1450.7 TIQQTFIDNDKK1454.7 VVTTNSILYDMAK 1571.7 KDIFGEVYTDSIGK 1593.7 QDPHAWLSLDNGIK 1818.9DVKPIYLNGEEGNKDK 1836.9 DKQDPHAWLSLDNGIK 1911.9 QYGITPGYIWEINTEK 2582.3LTDADVILYNGLNLETGNGWFEK 2710.2 KLTDADVILYNGLNLETGNGWFEK 2942.4NVGGDNVDIHSIVPVGQDPHEYEVKPK ¹Molecular weight as determined by SDS-PAGE.²The m/z value of a polypeptide fragment can be converted to mass bysubtracting 1 from the m/z value. Each mass includes a range of plus orminus 300 parts per million (ppm), or plus or minus 1 Da.

TABLE 3 Characteristics of polypeptides obtained from S. aureus isolateSAAV1. Approximate m/z value of polypeptide polypeptide molecular weightin fragments resulting from Predicted amino acid sequence designationkilodaltons (kDa)¹ trypsin digest² of the polypeptide fragment P33A 55783.4 LHSWLK 911.5 KLHSWLK 937.5 TYTFHLR 996.5 KFDGTGPFK 1025.5QAIGHMVNR 1039.4 NDQYWGEK 1178.5 GTDSLDKDSLK 1185.5 IYNSIDDAFK 1222.6DKYTVELNLK 1229.5 ISTLIDNVKVK 1346.6 AESLLDEAGWKK 1355.5 EQAEYLQAEFK1381.6 VMPAGETAFLSMK 1400.5 KGETNFAFTDDR 1419.6 FHDGTPFDADAVK 1422.6NVTDINFDMPTR 1483.6 EQAEYLQAEFKK 1547.7 FHDGTPFDADAVKK 1550.6NVTDINFDMPTRK 1559.7 LNINGETSDKIAER 1787.9 EILDGQEKPATQLFAK 1945.8DESADFNKNDQYWGEK 2239.0 VSFTQSQYELPFNEMQYK 2354.1 QIDDEGIFIPISHGSMTVVAPK2868.1 DIGDMNPHVYGGSMSAESMIYEPLVR P33B 55 895.4 FPYAANGR 904.5 ALLHASHR1045.5 EEGLAIKASK 1384.5 GEAYFVDNNSLR 1435.7 TIEADYVLVTVGR 1669.8RPNTDELGLEELGVK 1841.0 NAIIATGSRPIEIPNFK 2179.2 TSISNIYAIGDIVPGLPLAHK2546.2 FVEAQHSENLGVIAESVSLNFQK 2587.3 VVGDFPIETDTIVIGAGPGGYVAAIR P35 37699.4 FEYIK 729.4 DAWPLK 792.4 ASVVNFR 852.4 VYDQLSK 1008.4 LIDDLYEK1020.4 YKDAWPLK 1074.4 EKEAEDLLK 1083.5 LKPDLIVASK 1169.5 FEYIKNDLK1182.4 KTESEWTSSK 1184.4 YDDKVAAFQK 1278.5 IAPTVSTDTVFK 1558.7QVDNGKDIIQLTSK 1623.7 IVGQEPAPNLEEISK 1712.7 ESIPLMNADHIFVVK 1800.7IYAGGYAGEILNDLGFK 1956.8 IYAGGYAGEILNDLGFKR 2251.9 NNQVSDDLDEITWNLAGGYK3227.5 VVTLYQGATDVAVSLGVKPVGAVESWTQKPK P38 33 864.5 YIAQLEK 946.4QGTPEQMR 976.5 DKFNDIPK 1054.5 AMITSEGAFK 1146.5 FNDIPKEQR 1268.6HLLVETSVDKK 1322.5 TIQQTFIDNDK 1443.6 DIFGEVYTDSIGK 1450.6 TIQQTFIDNDKK1454.6 VVTTNSILYDMAK 1593.7 QDPHAWLSLDNGIK 1818.9 DVKPIYLNGEEGNKDK1836.8 DKQDPHAWLSLDNGIK 1911.9 QYGITPGYIWEINTEK 2942.4NVGGDNVDIHSIVPVGQDPHEYEVKPK ¹Molecular weight as determined by SDS-PAGE.²The m/z value of a polypeptide fragment can be converted to mass bysubtracting 1 from the m/z value. Each mass includes a range of plus orminus 300 parts per million (ppm) or plus or minus 1 Da.

TABLE 4 Characteristics of polypeptides obtained from S. aureus isolate2176. Approximate m/z value of polypeptide Polypeptide molecular weightin fragments resulting from Predicted amino acid sequence designationkilodaltons (kDa)¹ trypsin digest² of the polypeptide fragment P478 88736.35 IIGDYR 814.49 IFTDYR 942.42 IFTDYRK 945.36 TGNTPDGRK 974.40YAQVKPIR 984.27 QMQFFGAR 992.41 SMQPFGGIR 1087.31 EQQLDVISR 1097.31VSGYAVNFIK 1159.37 NHATAWQGFK 1261.37 LWEQVMQLSK 1289.46 AGVITGLPDAYGR1315.42 TSTFLDIYAER 1322.39 LREELSEQYR 1394.37 THNQGVFDAYSR 1417.52KAGVITGLPDAYGR 1426.36 IEMALHDTEIVR 1487.39 NHATAWQGFKNGR 1508.42AGEPFAPGANPMHGR 1513.52 VALYGVDFLMEEK 1543.43 YGFDLSRPAENFK 1571.50TSSIQYENDDIMR 1636.56 KAGEPFAPGANPMHGR 1859.80 DLETIVGVQTEKPFKR 1876.77TMATGIAGLSVAADSLSAIK 2042.57 MHDFNTMSTEMSEDVIR 2077.68YGNNDDRVDDIAVDLVER 2158.88 AGVITESEVQEIIDHFIMK 2284.90ETLIDAMEHPEEYPQLTIR 2575.08 FLHSLDNLGPAPEPNLTVLWSVR 2628.01SGAQVGPNFEGINSEVLEYDEVFK 2756.06 SGAQVGPNFEGINSEVLEYDEVFKK 3262.33VASTITSHDAGYLDKDLETIVGVQTEKPFK P479 80 625.27 HVDVR 736.26 IIGDYR 814.22IFTDYR 942.27 IFTDYRK 974.26 YAQVKPIR 984.18 QMQFFGAR 992.23 SMQPFGGIR1087.16 EQQLDVISR 1097.24 VSGYAVNFIK 1159.12 NHATAWQGFK 1243.14VDDIAVDLVER 1261.22 LWEQVMQLSK 1272.24 SLGKEPEDQNR 1277.18 DGISNTFSIVPK1289.21 AGVITGLPDAYGR 1315.19 TSTFLDIYAER 1322.21 LREELSEQYR 1394.16THNQGVFDAYSR 1417.32 KAGVITGLPDAYGR 1426.23 IEMALHDTEIVR 1487.19NHATAWQGFKNGR 1508.25 AGEPFAPGANPMHGR 1513.21 VALYGVDFLMEEK 1522.25KTHNQGVFDAYSR 1543.26 YGFDLSRPAENFK 1571.23 TSSIQYENDDIMR 1636.29KAGEPFAPGANPMHGR 1703.43 DLETIVGVQTEKPFK 1751.45 EAVQWLYLAYLAAIK 1859.53DLETIVGVQTEKPFKR 1876.50 TMATGIAGLSVAADSLSAIK 1936.37 NEEGLVVDFEIEGDFPK2042.43 MHDFNTMSTEMSEDVIR 2077.45 YGNNDDRVDDIAVDLVER 2158.57AGVITESEVQEIIDHFIMK 2284.61 ETLIDAMEHPEEYPQLTIR 2574.77FLHSLDNLGPAPEPNLTVLWSVR 2627.61 SGAQVGPNFEGINSEVLEYDEVFK 2755.70SGAQVGPNFEGINSEVLEYDEVFKK 2907.65 EFIQLNYTLYEGNDSFLAGPTEATSK 3261.91VASTITSHDAGYLDKDLETIVGVQTEKPFK 3421.02 TPDYNELFSGDPTWVTESIGGVGIDGRPLVTKP480 65 625.35 HVDVR 717.38 YSYER 733.42 LPDNFK 736.44 IIGDYR 814.33IFTDYR 853.31 YGNNDDR 942.33 IFTDYRK 944.39 ELKELGQK 974.52 YAQVKPIR984.36 QMQFFGAR 992.44 SMQPFGGIR 1049.44 TLLYAINGGK 1087.43 EQQLDVISR1097.51 VSGYAVNFIK 1159.52 NHATAWQGFK 1289.53 AGVITGLPDAYGR 1315.51TSTFLDIYAER 1322.46 LREELSEQYR 1394.50 THNQGVFDAYSR 1417.65KAGVITGLPDAYGR 1442.56 IEMALHDTEIVR + Oxidation (M) 1467.60VSGYAVNFIKLTR 1522.61 KTHNQGVFDAYSR 1524.55 AGEPFAPGANPMHGR + Oxidation(M) 1529.64 VALYGVDFLMEEK + Oxidation (M) 1543.62 YGFDLSRPAENFK 1652.68KAGEPFAPGANPMHGR + Oxidation (M) 1671.76 TSTFLDIYAERDLK 1766.76VDDIAVDLVERFMTK + Oxidation (M) 1876.86 TMATGIAGLSVAADSLSAIK 2077.93YGNNDDRVDDIAVDLVER 2225.07 DSEHTMSVLTITSNVVYGKK + Oxidation (M) 2575.33FLHSLDNLGPAPEPNLTVLWSVR 2628.25 SGAQVGPNFEGINSEVLEYDEVFK 2748.36NLTSMLDGYAMQCGHHLNINVFNR 2756.63 SGAQVGPNFEGINSEVLEYDEVFKK 3001.02DEKSGAQVGPNFEGINSEVLEYDEVFK 3420.75 TPDYNELFSGDPTWVTESIGGVGIDGRPLVTKP481 55 634.33 AKSNSK 883.24 TFYPEAR 1014.24 QFWGHLVK 1131.17 WIPLMMKGR1207.21 VINEEFEISK 1324.10 NEDWQLYTAGK 1360.28 TLLFGPFANVGPK 1386.31LDRPAIESSNER 1565.30 IDEGTDVNFGELTR 1584.34 EFINPLPHISYVR 1699.29EIEPDWNIHVYER 1744.36 EPPGTPPMTVPHLDTR 2046.52 QVTDYVFIGAGGGAIPLLQK2189.43 TFYPEARNEDWQLYTAGK 2806.58 HLGGFPISGQFLACTNPQVIEQHDAK P482 37699.28 FEYIK 729.26 DAWPLK 792.33 ASVVNFR 852.28 VYDQLSK 1008.30LIDDLYEK 1020.31 YKDAWPLK 1083.43 LKPDLIVASK 1278.36 IAPTVSTDTVFK1623.44 IVGQEPAPNLEEISK 1712.62 ESIPLMNADHIFVVK 1800.61IYAGGYAGEILNDLGFK 1956.77 IYAGGYAGEILNDLGFKR 2251.77NNQVSDDLDEITWNLAGGYK 3227.44 VVTLYQGATDVAVSLGVKPVGAVESWTQKPK P483 36646.50 DVWAR 672.41 KLNAVK 716.41 VDIVDR 725.61 IIKPVR 842.50 IAPTLSLK850.47 QNINSFK 1068.50 IGDYTSVGTR 1075.42 MIIMTDHAK + Oxidation (M)1185.53 KQPNLEEISK 1327.59 LKPDLIIADSSR 1343.58 VDIVDRDVWAR 1592.76LKPDLIIADSSRHK 2081.00 GPYLQLDTEHLADLNPER 2438.24 AGLLAHPNYSYVGQFLNELGFK2789.48 IVVLEYSFADALAALDVKPVGIADDGK 2917.60 IVVLEYSFADALAALDVKPVGIADDGKKP484 35 857.38 AAAIDLAGR 1022.23 NIEADTGMR + Oxidation (M) 1056.32VVDANIAAQR 1075.36 ADIDLPFER 1285.44 LVGGAGEETIIAR 1435.44 AMAVATEQEMKAR1632.50 HHTEVLENPDNISK 1813.65 VVEAESEVPLAMAEALR 1887.67VIETPFIAGVAMNGIEVK 2299.85 AGLALTTNQLESHYLAGGNVDR 2806.95TVLSKGLDSGTAFEILSIDIADVDISK 3337.42 AGLALTTNQLESHYLAGGNVDRVVDANIAAQRP485 33 625.28 ADYEK 864.28 YIAQLEK 946.23 QGTPEQMR 1045.26 ALEQAGKSLK1268.35 HLLVETSVDKK 1443.34 DIFGEVYTDSIGK 1450.40 TIQQTFIDNDKK 1454.37VVTTNSILYDMAK 1571.45 KDIFGEVYTDSIGK 1576.44 DVKPIYLNGEEGNK 1593.47QDPHAWLSLDNGIK 1819.59 DVKPIYLNGEEGNKDK 1836.62 DKQDPHAWLSLDNGIK 1911.66QYGITPGYIWEINTEK 2172.83 VIAVSKDVKPIYLNGEEGNK 2582.00LTDADVILYNGLNLETGNGWFEK 2942.26 NVGGDNVDIHSIVPVGQDPHEYEVKPK P486 32625.42 ADYEK 864.41 YIAQLEK 1268.48 HLLVETSVDKK 1443.49 DIFGEVYTDSIGK1450.53 TIQQTFIDNDKK 1454.61 VVTTNSILYDMAK 1576.64 DVKPIYLNGEEGNK1593.57 QDPHAWLSLDNGIK 1818.77 DVKPIYLNGEEGNKDK 1836.78 DKQDPHAWLSLDNGIK1911.81 QYGITPGYIWEINTEK 2582.18 LTDADVILYNGLNLETGNGWFEK 2942.32NVGGDNVDIHSIVPVGQDPHEYEVKPK ¹Molecular weight as determined by SDS-PAGE.²The m/z value of a polypeptide fragment can be converted to mass bysubtracting 1 from the m/z value. Each mass includes a range of plus orminus 400 parts per million (ppm) or 1 Dalton.

TABLE 5 Characteristics of polypeptides obtained from S. aureus bovineisolate 1477. Approximate m/z value of polypeptide polypeptide molecularweight in fragments resulting from Predicted amino acid sequencedesignation kilodaltons (kDa)¹ trypsin digest² of the polypeptidefragment P487 88 717.39 YSYER 736.52 IIGDYR 814.46 IFTDYR 942.46 IFTDYRK974.54 YAQVKPIR 984.41 QMQFFGAR 992.40 SMQPFGGIR 1087.49 EQQLDVISR1097.50 VSGYAVNFIK 1159.39 NHATAWQGFK 1261.45 LWEQVMQLSK 1272.50SLGKEPEDQNR 1277.50 DGISNTFSIVPK 1289.54 AGVITGLPDAYGR 1315.54TSTFLDIYAER 1322.53 LREELSEQYR 1394.50 THNQGVFDAYSR 1417.62KAGVITGLPDAYGR 1426.65 IEMALHDTEIVR 1508.59 AGEPFAPGANPMHGR 1522.61KTHNQGVFDAYSR 1543.68 YGFDLSRPAENFK 1877.74 TMATGIAGLSVAADSLSAIK 2077.86YGNNDDRVDDIAVDLVER 2159.08 AGVITESEVQEIIDHFIMK 2285.07ETLIDAMEHPEEYPQLTIR 2575.32 FLHSLDNLGPAPEPNLTVLWSVR 2628.24SGAQVGPNFEGINSEVLEYDEVFK 2756.41 SGAQVGPNFEGINSEVLEYDEVFKK 3262.68VASTITSHDAGYLDKDLETIVGVQTEKPFK P488 80 625.49 HVDVR 814.54 IFTDYR 942.66IFTDYRK 974.69 YAQVKPIR 984.59 MQFFGAR 992.55 SMQPFGGIR 1159.64NHATAWQGFK 1261.63 LWEQVMQLSK 1272.74 SLGKEPEDQNR 1277.69 DGISNTFSIVPK1289.76 AGVITGLPDAYGR 1315.73 TSTFLDIYAER 1322.72 SMQPFGGIRMAK 1394.73THNQGVFDAYSR 1417.86 KAGVITGLPDAYGR 1422.76 TLLYAINGGKDEK 1426.80IEMALHDTEIVR 1508.82 AGEPFAPGANPMHGR 1513.80 VALYGVDFLMEEK 1543.82YGFDLSRPAENFK 1571.82 TSSIQYENDDIMR 1703.99 DLETIVGVQTEKPFK 1860.23DLETIVGVQTEKPFKR 1877.07 TMATGIAGLSVAADSLSAIK 1937.09 NEEGLVVDFEIEGDFPK2078.13 YGNNDDRVDDIAVDLVER 2575.56 FLHSLDNLGPAPEPNLTVLWSVR 2628.30SGAQVGPNFEGINSEVLEYDEVFK 2908.63 EFIQLNYTLYEGNDSFLAGPTEATSK P489 65733.67 IVKFAR 944.71 ELKELGQK 974.79 YAQVKPIR 984.69 QMQFFGAR 1049.83TLLYAINGGK 1087.78 EQQLDVISR 1097.79 VSGYAVNFIK 1243.80 VDDIAVDLVER1272.82 SLGKEPEDQNR 1289.87 AGVITGLPDAYGR 1299.92 LPDNFKTYCAK 1315.83TSTFLDIYAER 1322.84 SMQPFGGIRMAK 1390.93 DQKGALSSLSSVAK 1394.84THNQGVFDAYSR 1577.94 VASTITSHDAGYLDK 1637.09 KAGEPFAPGANPMHGR 1704.16DLETIVGVQTEKPFK 2030.42 MSIKTSSIQYENDDIMR 2078.34 YGNNDDRVDDIAVDLVER2284.60 ETLIDAMEHPEEYPQLTIR 2575.77 FLHSLDNLGPAPEPNLTVLWSVR 2628.64SGAQVGPNFEGINSEVLEYDEVFK P490 55 883.81 TFYPEAR 1014.87 QFWGHLVK 1131.97WIPLMMKGR 1207.99 VINEEFEISK 1231.97 YSFDQVIMTK 1325.02 NEDWQLYTAGK1361.17 TLLFGPFANVGPK 1362.14 GREDNPGIMAASK + Oxidation (M) 1387.14LDRPAIESSNER 1481.24 NEDWQLYTAGKR 1566.28 IDEGTDVNFGELTR 1585.34EFINPLPHISYVR 1700.36 EIEPDWNIHVYER 1761.49 EPPGTPPMTVPHLDTR + Oxidation(M) 2047.67 QVTDYVFIGAGGGAIPLLQK 2208.82 VYGKEPPGTPPMTVPHLDTR +Oxidation (M) 2865.21 HLGGFPISGQFLACTNPQVIEQHDAK P492 36 857.57AAAIDLAGR 1056.59 VVDANIAAQR 1075.61 ADIDLPFER 1285.74 LVGGAGEETIIAR1632.95 HHTEVLENPDNISK 1814.09 VVEAESEVPLAMAEALR 2284.45AAAIDLAGRDVLEAVQMSVNPK + Oxidation (M) 2300.40 AGLALTTNQLESHYLAGGNVDR2807.80 TVLSKGLDSGTAFEILSIDIADVDISK P493 35 762.46 FVFHGR 964.39DGFNNIER 1363.56 GHVYNGISGGQFK 1443.56 YTPTSILYFNPK 1450.64QLAEDLQKHLGAK 1819.88 NHSEYVTDMRLIGIR + Oxidation (M) 1875.84DLPPMEQVFDTLDLDK 1941.00 IRPEDMHIMANIFLPK + Oxidation (M) 2081.10RIRPEDMHIMANIFLPK 2283.30 ISHLVLTRTGLYIIDSQLLK P495 32 ¹Molecular weightas determined by SDS-PAGE. ²The m/z value of a polypeptide fragment canbe converted to mass by subtracting 1 from the m/z value. Each massincludes a range of plus or minus 430 parts per million (ppm) or 1Dalton.

In yet another aspect, the present invention further includespolypeptides having similarity with an amino acid sequence. Thesimilarity is referred to as structural similarity and is generallydetermined by aligning the residues of the two amino acid sequences(i.e., a candidate amino acid sequence and a reference amino acidsequence) to optimize the number of identical amino acids along thelengths of their sequences; gaps in either or both sequences arepermitted in making the alignment in order to optimize the number ofidentical amino acids, although the amino acids in each sequence mustnonetheless remain in their proper order. Reference amino acid sequencesare disclosed in Tables 6, 7, 8, and 9. Two amino acid sequences can becompared using commercially available algorithms. Preferably, two aminoacid sequences are compared using the BLASTP program of the BLAST 2search algorithm, as described by Tatusova, et al., (FEMS Microbiol Lett1999, 174:247-250), and available through the World Wide Web, forinstance at the internet site maintained by the National Center forBiotechnology Information, National Institutes of Health. Preferably,the default values for all BLAST 2 search parameters are used, includingmatrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gapx_dropoff=50, expect=10, wordsize=3, and optionally, filter on. In thecomparison of two amino acid sequences using the BLAST search algorithm,structural similarity is referred to as “identities.” Preferably, acandidate amino acid sequence has at least 80% identity, at least 90%identity, at least 95% identity, at least 96% identity, at least 97%identity, at least 98% identity, or at least 99% identity to a referenceamino acid sequence. Preferably, the molecular weight of the candidateamino acid sequence and the reference amino acid sequence aresubstantially the same value. Preferably, the molecular weight of thecandidate amino acid sequence and the reference amino acid sequence isdetermined by SDS polyacrylamide gel electrophoresis. A candidatepolypeptide can be obtained by growth of a microbe under low metalconditions and the subsequent isolation of a polypeptide by theprocedures disclosed herein.

Typically, a candidate amino acid sequence having structural similarityto a reference amino acid sequence has immunogenic activity, protectiveimmunogenic activity, seroactive activity, immunoregulatory activity, ora combination thereof.

TABLE 6 S. aureus ATCC isolate 19636. NCBI sequence identifier ofpolypeptide identified by the Molecular weight of computer algorithm ashaving reference polypeptide best match to mass fingerprint Sequence ID(kDa)¹ of reference polypeptide Number 88 49243545 418 55 81762012 41938 82750440 420 37 49243435 421 36 57286380 422 35 49245508 423 3349243946 424 ¹Molecular weight as determined by SDS-PAGE.

TABLE 7 S. aureus SAAV1. NCBI sequence identifier of polypeptideidentified by the Molecular weight of computer algorithm as havingreference polypeptide best match to mass fingerprint (kDa)¹ of referencepolypeptide 55 57286470 55 48874 37 49243435 33 49243946 ¹Molecularweight as determined by SDS-PAGE.

TABLE 8 S. aureus 2176. NCBI sequence identifier of polypeptideidentified by the Molecular weight of computer algorithm as havingreference polypeptide best match to mass fingerprint (kDa)¹ of referencepolypeptide 88 57285406 80 57285406 65 57285406 55 57286528 37 4948235836 57286380 35 15927153 33 57285658 32 57285658 ¹Molecular weight asdetermined by SDS-PAGE.

TABLE 9 S. aureus 1477. NCBI sequence identifier of polypeptideidentified by the Molecular weight of computer algorithm as havingreference polypeptide best match to mass fingerprint (kDa)¹ of referencepolypeptide 88 49482458 80 57285406 65 57285406 55 57286528 36 1592715335 49484031 ¹Molecular weight as determined by SDS-PAGE.

The polypeptides expressed by a reference microbe and referred to aboveby molecular weight can be obtained by growth of the reference microbeunder low metal conditions and the subsequent isolation of a polypeptideby the processes disclosed herein. A candidate polypeptide is isolatablefrom a microbe, preferably a gram positive microbe, more preferably, amember of the family Micrococcaceae, preferably, Staphylococcus spp.,more preferably, Staphylococcus aureus. Other gram positive microbesinclude Corynebacterium spp., Erysipelothrix spp., Mycobacterium spp.,and Erysipelothrix spp. A candidate polypeptide may also be producedusing recombinant, enzymatic, or chemical techniques.

Also provided by the present invention are whole cell preparations of amicrobe, where the microbe expresses one or more of the polypeptides ofthe present invention. The cells present in a whole cell preparation arepreferably inactivated such that the cells cannot replicate, but theimmunogenic activity of the polypeptides of the present inventionexpressed by the microbe is maintained. Typically, the cells are killedby exposure to agents such as glutaraldehyde, formalin, or formaldehyde.

Compositions

A composition of the present invention may include at least onepolypeptide described herein, or a number of polypeptides that is aninteger greater than 1 (e.g., at least 2, at least 3, at least 4). Forexample, a composition can include 2, 3, 4, 5, or more isolated metalregulated polypeptides having molecular weights of 88 kDa, 55 kDa, 38kDa, 37 kDa, 36 kDa, 35 kDa, 33 kDa, or any subset or combinationthereof. A composition can include polypeptides isolatable from 1microbe, or can be isolatable from a combination of 2 or more microbes.For instance, a composition can include polypeptides isolatable from 2or more Staphyloccocus spp., or from a Staphyloccocus spp. and adifferent microbe that is not a member of the genus Staphyloccocus. Thepresent invention also provides compositions including a whole cellpreparation, where the whole cell expresses one or more of thepolypeptides of the present invention. For instance, the whole cell canbe a Staphyloccocus spp. In some aspects, a composition can includewhole preparations from 2, 3, 4, 5, or 6 strains.

Optionally, a polypeptide of the present invention can be covalentlybound or conjugated to a carrier polypeptide to improve theimmunological properties of the polypeptide. Useful carrier polypeptidesare known in the art. The chemical coupling of polypeptides of thepresent invention can be carried out using known and routine methods.For instance, various homobifunctional and/or heterobifunctionalcross-linker reagents such as bis(sulfosuccinimidyl) suberate,bis(diazobenzidine), dimethyl adipimidate, dimethyl pimelimidate,dimethyl superimidate, disuccinimidyl suberate, glutaraldehyde,m-maleimidobenzoyl-N-hydroxysuccinimide,sulfo-m-maleimidobenzoyl-N-hydroxysuccinimide, sulfosuccinimidyl4-(N-maleimidomethyl)cycloheane-1-carboxylate, sulfosuccinimidyl4-(p-maleimido-phenyl) butyrate and (1-ethyl-3-(dimethyl-aminopropyl)carbodimide can be used (see, for instance, Harlow and Lane, Antibodies,A Laboratory Manual, generally and Chapter 5, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., N.Y. (1988)).

The compositions of the present invention optionally further include apharmaceutically acceptable carrier. “Pharmaceutically acceptable”refers to a diluent, carrier, excipient, salt, etc, that is compatiblewith the other ingredients of the composition, and not deleterious tothe recipient thereof. Typically, the composition includes apharmaceutically acceptable carrier when the composition is used asdescribed herein. The compositions of the present invention may beformulated in pharmaceutical preparations in a variety of forms adaptedto the chosen route of administration, including routes suitable forstimulating an immune response to an antigen. Thus, a composition of thepresent invention can be administered via known routes including, forexample, oral; parental including intradermal, transcutaneous andsubcutaneous; intramuscular, intravenous, intraperitoneal, etc. andtopically, such as, intranasal, intrapulmonary, intramammary,intravaginal, intrauterine, intradermal, transcutaneous and rectally,etc. It is foreseen that a composition can be administered to a mucosalsurface, such as by administration to the nasal or respiratory mucosa(e.g. spray or aerosol), in order to stimulate mucosal immunity, such asproduction of secretory IgA antibodies, throughout the animal's body.

A composition of the present invention can also be administered via asustained or delayed release implant. Implants suitable for useaccording to the invention are known and include, for example, thosedisclosed in Emery and Straub (WO 01/37810 (2001)), and Emery et al.,(WO 96/01620 (1996)). Implants can be produced at sizes small enough tobe administered by aerosol or spray. Implants also include nanospheresand microspheres.

A composition of the present invention may be administered in an amountsufficient to treat certain conditions as described herein. The amountof polypeptides or whole cells present in a composition of the presentinvention can vary. For instance, the dosage of polypeptides can bebetween 0.01 micrograms (μg) and 300 mg, typically between 0.1 mg and 10mg. When the composition is a whole cell preparation, the cells can bepresent at a concentration of, for instance, 10² bacteria/ml, 10³bacteria/ml, 10⁴ bacteria/ml, 10⁵ bacteria/ml, 10⁶ bacteria/ml, 10⁷bacteria/ml, 10⁸ bacteria/ml, or 10⁹ bacteria/ml. For an injectablecomposition (e.g. subcutaneous, intramuscular, etc.) the polypeptidesmay be present in the composition in an amount such that the totalvolume of the composition administered is 0.5 ml to 5.0 ml, typically1.0-2.0 ml. When the composition is a whole cell preparation, the cellsare preferably present in the composition in an amount that the totalvolume of the composition administered is 0.5 ml to 5.0 ml, typically1.0-2.0 ml. The amount administered will vary depending on variousfactors including, but not limited to, the specific polypeptides chosen,the weight, physical condition and age of the animal, and the route ofadministration. Thus, the absolute weight of the polypeptide included ina given unit dosage form can vary widely, and depends upon factors suchas the species, age, weight and physical condition of the animal, aswell as the method of administration. Such factors can be determined byone of skill in the art. Other examples of dosages suitable for theinvention are disclosed in Emery et al., (U.S. Pat. No. 6,027,736).

The formulations may be conveniently presented in unit dosage form andmay be prepared by methods well known in the art of pharmacy. Methods ofpreparing a composition with a pharmaceutically acceptable carrierinclude the step of bringing the active compound (e.g., a polypeptide orwhole cell of the present invention) into association with a carrierthat constitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing theactive compound into association with a liquid carrier, a finely dividedsolid carrier, or both, and then, if necessary, shaping the product intothe desired formulations.

A composition including a pharmaceutically acceptable carrier can alsoinclude an adjuvant. An “adjuvant” refers to an agent that can act in anonspecific manner to enhance an immune response to a particularantigen, thus potentially reducing the quantity of antigen necessary inany given immunizing composition, and/or the frequency of injectionnecessary in order to generate an adequate immune response to theantigen of interest. Adjuvants may include, for example, IL-1, IL-2,emulsifiers, muramyl dipeptides, dimethyl dioctadecyl ammonium bromide(DDA), pyridine, aluminum hydroxide, oils, saponins, alpha-tocopherol,polysaccharides, emulsified paraffins (including, for instance, thoseavailable from under the tradename EMULSIGEN from MVP Laboratories,Ralston, Nebr.), ISA-70, R1B1 and other substances known in the art. Itis expected that polypeptides of the present invention will haveimmunoregulatory activity and that such polypeptides may be used asadjuvants that directly act as T and/or B cell activators or act onspecific cell types that enhance the synthesis of various cytokines oractivate intracellular signaling pathways. Such polypeptides areexpected to augment the immune response to increase the protective indexof the existing composition.

In another embodiment, a composition of the invention including apharmaceutically acceptable carrier can include a biological responsemodifier, such as, for example, IL-2, IL-4 and/or IL-6, TNF, IFN-alpha,IFN-gamma, and other cytokines that effect immune cells. An immunizingcomposition can also include other components known in the art such asan antibiotic, a preservative, an anti-oxidant, or a chelating agent.

Methods of Making

The present invention also provides methods for obtaining thepolypeptides described herein. The polypeptides and whole cells of thepresent invention are isolatable from a member of the familyMicrococcaceae, preferably, Staphylococcus spp., more preferably,Staphylococcus aureus. Other gram positive microbes from whichpolypeptides can be isolated include Cornebacterium spp., Erysipelothrixspp., Mycobacterium spp., and Erysipelothrix spp. Microbes useful forobtaining polypeptides of the present invention and making whole cellpreparations are commercially available from a depository such asAmerican Type Culture Collection (ATCC). In addition, such microbes arereadily obtainable by techniques routine and known to the art. Themicrobes may be derived from an infected animal as a field isolate, andused to obtain polypeptides and/or whole cell preparations of thepresent invention, or stored for future use, for example, in a frozenrepository at −20° C. to −95° C., or −40° C. to −50° C., inbacteriological media containing 20% glycerol, and other like media.

When a polypeptide of the present invention is to be obtained from amicrobe, the microbe can be incubated under low metal conditions. Asused herein, the phrase “low metal conditions” refers to an environment,typically bacteriological media, which contains amounts of a free metalthat cause a microbe to express metal regulated polypeptides at adetectable level. As used herein, the phrase “high metal conditions”refers to an environment that contains amounts of a free metal thatcause a microbe to either not express one or more of the metal regulatedpolypeptides described herein at a detectable level, or to decreaseexpression of such a polypeptide. Metals are those present in theperiodic table under Groups 1 through 17 (TUPAC notation; also referredto as Groups I-A, II-A, III-B, IV-B, V-B, VI-B, VII-B, VIII, I-B, II-B,III-A, IV-A, V-A, VI-A, and VII-A, respectively, under CAS notation).Preferably, metals are those in Groups 2 through 12, more preferably,Groups 3-12. Even more preferably, the metal is iron, zinc, copper,magnesium, nickel, cobalt, manganese, molybdenum, or selenium, mostpreferably, iron.

Low metal conditions are generally the result of the addition of a metalchelating compound to a bacteriological medium, the use of abacteriological medium that contains low amounts of a metal, or thecombination thereof. High metal conditions are generally present when achelator is not present in the medium, a metal is added to the medium,or the combination thereof. Examples of metal chelators include naturaland synthetic compounds. Examples of natural compounds include plantphenolic compounds, such as flavenoids. Examples of flavenoids includethe copper chelators catechin and naringenin, and the iron chelatorsmyricetin and quercetin. Examples of synthetic copper chelators include,for instance, tetrathiomolybdate, and examples of synthetic zincchelators include, for instance, N,N,N′,N′-Tetrakis(2-pyridylmethyl)-ethylene diamine. Examples of synthetic iron chelatorsinclude 2,2′-dipyridyl (also referred to in the art as α,α′-bipyridyl),8-hydroxyquinoline, ethylenediamine-di-O-hydroxyphenylacetic acid(EDDHA), desferrioxamine methanesulphonate (desferol), transferrin,lactoferrin, ovotransferrin, biological siderophores, such as, thecatecholates and hydroxamates, and citrate. An example of a generaldivalent cation chelator is Chelex® resin. Preferably, 2,2′-dipyridyl isused for the chelation of iron. Typically, 2,2′-dipyridyl is added tothe media at a concentration of at least 300 micrograms/milliliter(μg/ml), at least 600 μg/ml, or at least 900 μg/ml. High levels of2,2′-dipyridyl can be 1200 μg/ml, 1500 μg/ml, or 1800 μg/ml.

The S. aureus genome encodes three Fur homologs: Fur, PerR, and Zur.While the Zur and PerR proteins appear to be primarily involved inregulating zinc homeostasis and peroxide stress genes, respectively, theFur protein has been demonstrated to regulate several iron-siderophoreuptake systems in response to iron limitation. The Fur protein alsoplays a role in oxidative stress resistance and virulence. It isexpected that a gram positive organism, preferably, an S. aureus, with amutation in a fur gene will result in the constitutive expression ofmany, if not all, of the metal regulated polypeptides of the presentinvention. The production of a fur mutation in a gram positive,preferably, an S. aureus, can be produced using routine methodsincluding, for instance, transposon, chemical, or site-directedmutagenesis useful for generating gene knock-out mutations in grampositive bacteria.

The medium used to incubate the microbe and the volume of media used toincubate the microbe can vary. When a microbe is being evaluated for theability to produce one or more of the polypeptides described herein, themicrobe can be grown in a suitable volume, for instance, 10 millilitersto 1 liter of medium. When a microbe is being grown to obtainpolypeptides for use in, for instance, administration to animals, themicrobe may be grown in a fermentor to allow the isolation of largeramounts of polypeptides. Methods for growing microbes in a fermentor areroutine and known to the art. The conditions used for growing a microbepreferably include a metal chelator, more preferably an iron chelator,for instance 2,2′-dipyridyl, a pH of between 6.5 and 7.5, preferablybetween 6.9 and 7.1, and a temperature of 37° C.

In some aspects of the invention, a microbe may be harvested aftergrowth. Harvesting includes concentrating the microbe into a smallervolume and suspending in a media different than the growth media.Methods for concentrating a microbe are routine and known in the art,and include, for example, filtration or centrifugation. Typically, theconcentrated microbe is suspended in an appropriate buffer. An exampleof a buffer that can be used contains Tris-base (7.3 grams/liter), at apH of 8.5. Optionally, the final buffer also minimizes proteolyticdegradation. This can be accomplished by having the final buffer at a pHof greater than 8.0, preferably, at least 8.5, and/or including one ormore proteinase inhibitors (e.g., phenylmethanesulfonyl fluoride).Optionally and preferably, the concentrated microbe is frozen at −20° C.or below until disrupted.

When the microbe is to be used as a whole cell preparation, theharvested cells may be processed using routine and known methods toinactivate the cells. Alternatively, when a microbe is to be used toprepare polypeptides of the present invention, the microbe may bedisrupted using chemical, physical, or mechanical methods routine andknown to the art, including, for example, boiling, french press,sonication, digestion of peptidoglycan (for instance, by digestion withlysozyme), or homogenization. An example of a suitable device useful forhomogenization is a model C500-B Avestin Homogenizer, (Avestin Inc,Ottawa Canada). As used herein, “disruption” refers to the breaking upof the cell. Disruption of a microbe can be measured by methods that areroutine and known to the art, including, for instance, changes inoptical density. Typically, a microbe is subjected to disruption untilthe percent transmittance is increased by 20% when a 1:100 dilution ismeasured. When physical or mechanical methods are used, the temperatureduring disruption is typically kept low, preferably at 4° C., to furtherminimize proteolytic degradation. When chemical methods are used thetemperature may be increased to optimize for the cell disruption. Acombination of chemical, physical, and mechanical methods may also beused to solubilize the cell wall of microbe. As used herein, the term“solubilize” refers to dissolving cellular materials (e.g.,polypeptides, nucleic acids, carbohydrates) into the aqueous phase ofthe buffer in which the microbe was disrupted, and the formation ofaggregates of insoluble cellular materials. Without intending to belimited by theory, the conditions for solubilization are believed toresult in the aggregation of polypeptides of the present invention intoinsoluble aggregates that are large enough to allow easy isolation by,for instance, centrifugation.

The insoluble aggregates that include one or more of the polypeptides ofthe present invention may be isolated by methods that are routine andknown to the art. Preferably, the insoluble aggregates are isolated bycentrifugation. Typically, centrifugation of polypeptides, such asmembrane polypeptides, can be accomplished by centrifugal forces of100,000×g. The use of such centrifugal forces requires the use ofultracentrifuges, and scale-up to process large volumes of sample isoften difficult and not economical with these types of centrifuges. Themethods described herein provide for the production of insolubleaggregates large enough to allow the use of continuous flow centrifuges,for instance T-1 Sharples (Alfa Laval Separations, Warminster, Pa.),which can be used with a flow rate of 250 ml/minute at 17 psi at acentrifugal force of 46,000×g to 60,000×g. Other large scale centrifugescan be used, such as the tubular bowl, chamber, and disc configurations.Such centrifuges are routinely used and known in the art, and arecommercially available from such manufactures as Pennwalt, Westfalia andalpha-Laval.

The final harvested proteins are washed and/or dialyzed against anappropriate buffer using methods known in the art, for instancediafiltration, precipitation, hydrophobic chromatography, ion-exchangechromatography, or affinity chromatography, or ultra filtration andwashing the polypeptides, for instance, in alcohol, by diafiltration.After isolation, the polypeptides suspended in buffer and stored at lowtemperature, for instance, −20° C. or below.

In those aspects of the present invention where a whole cell preparationis to be made, after growth a microbe can be killed with the addition ofan agent such as glutaraldehyde, formalin, or formaldehyde, at aconcentration sufficient to inactivate the cells in the culture. Forinstance, formalin can be added at a concentration of 0.3% (vol:vol).After a period of time sufficient to inactivate the cells, the cells canbe harvested by, for instance, diafiltration and/or centrifugation, andwashed.

Methods of Use

An aspect of the present invention is further directed to methods ofusing the compositions of the present invention. The methods includeadministering to an animal an effective amount of a composition of thepresent invention. The animal can be, for instance, avian (including,for instance, chickens or turkeys), bovine (including, for instance,cattle), caprine (including, for instance, goats), ovine (including, forinstance, sheep), porcine (including, for instance, swine), bison(including, for instance, buffalo), equine (including, for instance,horses), a companion animal (including, for instance, dogs or cats),members of the family Cervidae (including, for instance, deer, elk,moose, caribou and reindeer), or human.

In some aspects, the methods may further include additionaladministrations (e.g., one or more booster administrations) of thecomposition to the animal to enhance or stimulate a secondary immuneresponse. A booster can be administered at a time after the firstadministration, for instance, 1 to 8 weeks, preferably 2 to 4 weeks,after the first administration of the composition. Subsequent boosterscan be administered one, two, three, four, or more times annually.Without intending to be limited by theory, it is expected that in someaspects of the present invention annual boosters will not be necessary,as an animal will be challenged in the field by exposure to microbesexpressing polypeptides present in the compositions having epitopes thatare identical to or structurally related to epitopes present onpolypeptides of the composition administered to the animal.

In one aspect, the invention is directed to methods for makingantibodies, for instance by inducing the production of antibody in ananimal, or by recombinant techniques. The antibody produced includesantibody that specifically binds at least one polypeptide present in thecomposition. In this aspect of the invention, an “effective amount” isan amount effective to result in the production of antibody in theanimal. Methods for determining whether an animal has producedantibodies that specifically bind polypeptides present in a compositionof the present invention can be determined as described herein. Thepresent invention further includes antibody that specifically bind to apolypeptide of the present invention, and compositions including suchantibodies. The method may be used to produce antibody that specificallybinds polypeptides expressed by a microbe other than the microbe fromwhich the polypeptides of the composition were isolated. As used herein,an antibody that can “specifically bind” a polypeptide is an antibodythat interacts with the epitope of the antigen that induced thesynthesis of the antibody, or interacts with a structurally relatedepitope. At least some of the polypeptides present in the compositionsof the present invention typically include epitopes that are conservedin the polypeptides of different species and different genera ofmicrobes. Accordingly, antibody produced using a composition derivedfrom one microbe is expected to bind to polypeptides expressed by othermicrobes and provide broad spectrum protection against gram positiveorganisms. Examples of gram positive microbes to which the antibody mayspecifically bind are Micrococcaceae, preferably, Staphylococcus spp.,more preferably, Staphylococcus aureus; members of the familyStreptococcaceae, preferably, Streptococcus pyrogenes, Streptococcuspneumoniae, Streptococcus agalactiae, Streptococcus uberis,Streptococcus bovis, Streptococcus equi, or Streptococcus dysgalactiae;and Bacillus spp., Clostridium spp., Corynebacterium spp., Enterococcusspp., Erysipelothrix spp., Listeria spp., Micrococcus spp., andMycobacterium spp., Kytococcus spp., and Erysipelothrix spp.

The present invention is also directed to the use of such antibody totarget a microbe expressing a polypeptide of the present invention or apolypeptide having an epitope structurally related to an epitope presenton a polypeptide of the present invention. A compound can be covalentlybound to an antibody, where the compound can be, for instance, a toxin.Likewise, such compounds can be covalently bound to a bacterialsiderophore to target the microbe. The chemical coupling or conjugationof an antibody of the present invention, or a portion thereof (such asan Fab fragment), can be carried out using known and routine methods. Inone aspect the invention is also directed to treating an infection in ananimal, including a human, caused by a gram positive microbe, preferablyby a member of the family Micrococcaceae, preferably, Staphylococcusspp., more preferably, S. aureus; members of the familyStreptococcaceae, preferably, Streptococcus pyogenes, Streptococcuspneumoniae, Streptococcus agalactiae, Streptococcus uberis,Streptococcus bovis, Streptococcus equi, or Streptococcus dysgalactiae;Bacillus spp., Clostridium spp., Corynebacterium spp., Enterococcusspp., Erysipelothrix spp., Kytococcus spp., Listeria spp., Micrococcusspp., Mycobacterium spp., and Erysipelothrix spp. As used herein, theterm “infection” refers to the presence of a gram positive microbe in ananimal's body, which may or may not be clinically apparent. An animalwith an infection by a member of the genus Staphylococcus that is notclinically apparent is often referred to as an asymptomatic carrier. Themethod includes administering an effective amount of the composition ofthe present invention to an animal having an infection caused by a grampositive microbe, and determining whether the number of microbes causingthe infection has decreased. Methods for determining whether aninfection is caused by a gram positive microbe are routine and known inthe art, as are methods for determining whether the infection hasdecreased.

In another aspect, the present invention is directed to methods fortreating one or more symptoms of certain conditions in an animal thatmay be caused by infection by a gram positive microbe, preferably by amember of the family Micrococcaceae, preferably, Staphylococcus spp.,more preferably, S. aureus; members of the family Streptoococcaceae,preferably, Streptococcus pyogenes, Streptococcus pneumoniae,Streptococcus agalactiae, Streptococcus uberis, Streptococcus bovis,Streptococcus equi, or Streptococcus dysgalactiae; Bacillus spp.,Clostridium spp., Corynebacterium spp., Enterococcus spp.,Erysipelothrix spp., Kytococcus spp., Listeria spp., Micrococcus spp.,Mycobacterium spp., and Erysipelothrix spp. The method includesadministering an effective amount of a composition of the presentinvention to an animal having or at risk of having a condition, orsymptoms of a condition, and determining whether at least one symptom ofthe condition is changed, preferably, reduced. Examples of conditionscaused by microbial infections include, for instance, mastitis,septicemia, pneumonia, meningoencephalitis, lymphangitis, dermatitis,genital tract infections, strangles, metritis, perinatal disease,pituitary abscesses, arthritis, bursitis, orchitis, cystitis andpyelonephritis, caseous lymphadenitis, tuberculosis, ulcerativelymphangitis, listeriosis, erysipelas, laminitis, anthrax, tyzzer'sdisease, tetanus, botulism, enteritis, malignant edema, braxy, bacillaryhemoglobinuria, enterotoxemia, necrotic skin lesions, and nosocomialinfections. Examples of conditions caused by S. aureus also include, forinstance, botryomycosis in horses, purulent synovitis and osteomyelitisin poultry, abortions in swine, and tick pyemia in lambs. Examples ofconditions caused by Streptococcus spp. also include, for instance, sorethroat, scarlet fever, impetigo, ulcerative endocarditis, rheumaticfever and post streptococcal glomerulonephritis cervicitis in humans,cervicitis in equine and swine, and meningitis and jowl abscesses inswine.

Treatment of symptoms associated with these conditions can beprophylactic or, alternatively, can be initiated after the developmentof a condition described herein. As used herein, the term “symptom”refers to objective evidence in a subject of a condition caused byinfection by a microbe. Symptoms associated with conditions referred toherein and the evaluations of such symptoms are routine and known in theart. Treatment that is prophylactic, for instance, initiated before asubject manifests symptoms of a condition caused by a microbe, isreferred to herein as treatment of a subject that is “at risk” ofdeveloping the condition. Typically, an animal “at risk” of developing acondition is an animal present in an area where animals having thecondition have been diagnosed and/or is likely to be exposed to amicrobe causing the condition. Accordingly, administration of acomposition can be performed before, during, or after the occurrence ofthe conditions described herein. Treatment initiated after thedevelopment of a condition may result in decreasing the severity of thesymptoms of one of the conditions, or completely removing the symptoms.In this aspect of the invention, an “effective amount” is an amounteffective to prevent the manifestation of symptoms of a disease,decrease the severity of the symptoms of a disease, and/or completelyremove the symptoms. The successful treatment of a gram positivemicrobial infection in an animal is disclosed in Example 5, whichdemonstrates the protection against disease caused by S. aureus in mousemodels by administering a composition of the present invention. Thesemouse models are a commonly accepted model for the study of humandisease caused by these microbes. The successful treatment of a grampositive microbial infection in an animal is also disclosed in Examples10-12, which demonstrates the protection against disease caused by S.aureus in cows by administering a composition of the present invention.

The present invention also provides methods for decreasing colonizationby gram positive microbes, for instance blocking the attachment sites ofgram positive microbe, including tissues of the skeletal system (forinstance, bones, cartilage, tendons and ligaments), muscular system,(for instance, skeletal and smooth muscles), circulatory system (forinstance, heart, blood vessels, capillaries and blood), nervous system(for instance, brain, spinal cord, and peripheral nerves), respiratorysystem (for instance, nose, trachea lungs, bronchi, bronchioceles,alveoli), digestive system (for instance, mouth, salivary glandsoesophagus liver stomach large and small intestine), excretory system(for instance, kidneys, ureters, bladder and urethra), endocrine system(for instance, hypothalamus, pituitary, thyroid, pancreas and adrenalglands), reproductive system (for instance, ovaries, oviduct, uterus,vagina, mammary glands, testes, and seminal vesicles), lymphatic/immunesystems (for instance, lymph, lymph nodes and vessels, mononuclear orwhite blood cells, such as macrophages, neutrophils, monocytes,eosinophils, basophils, lymphocytes t- and b-cells), and specific celllineages (for instance, precursor cells, epithelial cells, stem cells),and the like. Preferably, the gram positive microbe is a member of thefamily Micrococcaceae, preferably, Staphylococcus spp., more preferably,S. aureus; a member of the family Streptooccaceae, preferably,Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcusagalactiae, Streptococcus uberis, Streptococcus bovis, Streptococcusequi, or Streptococcus dysgalactiae; Bacillus spp., Clostridium spp.,Corynebacterium spp., Enterococus spp., Erysipelothrix spp., Kytococcusspp., Listeria spp., Micrococcus spp., Mycobacterium spp., andErysipelothrix spp. The method includes administering an effectiveamount of a composition of the present invention to an animal colonizedby, or at risk of being colonized by, a gram positive microbe. In thisaspect of the invention, an “effective amount” is an amount sufficientto decrease colonization of the animal by the microbe. Methods forevaluating the colonization of an animal by a microbe are routine andknown in the art. For instance, colonization of an animal's intestinaltract by a microbe can be determined by measuring the presence of themicrobe in the animal's feces. It is expected that decreasing thecolonization of an animal by a microbe will reduce transmission of themicrobe to humans.

A composition of the invention can be used to provide for active orpassive immunization against bacterial infection. Generally, thecomposition can be administered to an animal to provide activeimmunization. However, the composition can also be used to induceproduction of immune products, such as antibodies, which can becollected from the producing animal and administered to another animalto provide passive immunity. Immune components, such as antibodies, canbe collected to prepare compositions (preferably containing antibody)from serum, plasma, blood, colostrum, etc. for passive immunizationtherapies. Antibody compositions including monoclonal antibodies and/oranti-idiotypes can also be prepared using known methods. Chimericantibodies include human-derived constant regions of both heavy andlight chains and murine-derived variable regions that areantigen-specific (Morrison et al., Proc. Natl. Acad. Sci. USA, 1984,81(21):6851-5; LoBuglio et al., Proc. Natl. Acad. Sci. USA, 1989,86(11):4220-4; Boulianne et al., Nature, 1984, 312(5995):643-6).Humanized antibodies substitute the murine constant and framework (FR)(of the variable region) with the human counterparts (Jones et al.,Nature, 1986, 321(6069):522-5; Riechmann et al., Nature, 1988,332(6162):323-7; Verhoeyen et al., Science, 1988, 239(4847):1534-6;Queen et al., Proc. Natl. Acad. Sci. USA, 1989, 86(24):10029-33;Daugherty et al., Nucleic Acids Res., 1991, 19(9): 2471-6).Alternatively, certain mouse strains can be used that have beengenetically engineered to produce antibodies that are almost completelyof human origin; following immunization the B cells of these mice areharvested and immortalized for the production of human monoclonalantibodies (Bruggeman and Taussig, Curr. Opin. Biotechnol., 1997,8(4):455-8; Lonberg and Huszar, Int. Rev. Immunol., 1995; 13(1):65-93;Lonberg et al., Nature, 1994, 368:856-9; Taylor et al., Nucleic AcidsRes., 1992, 20:6287-95). Passive antibody compositions and fragmentsthereof, e.g., scFv, Fab, F(ab′)₂ or Fv or other modified forms thereof,may be administered to a recipient in the form of serum, plasma, blood,colostrum, and the like. However, the antibodies may also be isolatedfrom serum, plasma, blood, colostrum, and the like, using known methodsfor later use in a concentrated or reconstituted form such as, forinstance, lavage solutions, impregnated dressings and/or topical agentsand the like. Passive immunization preparations may be particularlyadvantageous for the treatment of acute systemic illness, or passiveimmunization of young animals that failed to receive adequate levels ofpassive immunity through maternal colostrum. Antibodies useful forpassive immunization may also be useful to conjugate to various drugs orantibiotics that could be directly targeted to bacteria expressingduring a systemic or localized infection a polypeptide of the presentinvention or a polypeptide having an epitope structurally related to anepitope present on a polypeptide of the present invention.

Animal models, in particular mouse models, are available forexperimentally evaluating the compositions of the present invention.These mouse models are commonly accepted models for the study of humandisease caused by members of the genus Staphylococcus, and S. aureus inparticular. In those cases where a members of the genus Staphylococcuscauses disease in an animal, for instance a cow, the natural host can beused to experimentally evaluate the compositions of the presentinvention.

Another aspect of the present invention provides methods for detectingantibody that specifically binds polypeptides of the present invention.These methods are useful in, for instance, detecting whether an animalhas antibody that specifically binds polypeptides of the presentinvention, and diagnosing whether an animal may have a condition causedby a microbe expressing polypeptides described herein, or expressingpolypeptides that share epitopes with the polypeptides described herein.Such diagnostic systems may be in kit form. The methods includecontacting an antibody with a preparation that include a polypeptide ofthe present invention to result in a mixture. The antibody may bepresent in a biological sample, for instance, blood, milk, or colostrum.The method further includes incubating the mixture under conditions toallow the antibody to specifically bind the polypeptide to form apolypeptide:antibody complex. As used herein, the termpolypeptide:antibody complex refers to the complex that results when anantibody specifically binds to a polypeptide. The preparation thatincludes the polypeptides of the present invention may also includereagents, for instance a buffer, that provide conditions appropriate forthe formation of the polypeptide:antibody complex. Thepolypeptide:antibody complex is then detected. The detection ofantibodies is known in the art and can include, for instance,immunofluorescence or peroxidase. The methods for detecting the presenceof antibodies that specifically bind to polypeptides of the presentinvention can be used in various formats that have been used to detectantibody, including radioimmunoassay and enzyme-linked immunosorbentassay.

The present invention also provides a kit for detecting antibody thatspecifically binds polypeptides of the present invention. The antibodydetected may be obtained from an animal suspected to have an infectioncaused by a gram positive microbe, more preferably, a member of thefamily Micrococcaceae, preferably, Staphylococcus spp., more preferably,S. aureus; Streptococcus spp., Bacillus spp., Clostridium spp.,Corynebacterium spp., Enterococus spp., Erysipelothrix spp., Kytococcusspp., Listeria spp., Micrococcus spp., Mycobacteriun spp., andErysipelothrix spp.

The kit includes at least one of the polypeptides of the presentinvention, or a number of polypeptides that is an integer greater than 1(e.g., at least 2, at least 3, etc.), in a suitable packaging materialin an amount sufficient for at least one assay. Optionally, otherreagents such as buffers and solutions needed to practice the inventionare also included. For instance, a kit may also include a reagent topermit detection of an antibody that specifically binds to a polypeptideof the present invention, such as a detectably labeled secondaryantibody designed to specifically bind to an antibody obtained from ananimal. Instructions for use of the packaged polypeptides are alsotypically included. As used herein, the phrase “packaging material”refers to one or more physical structures used to house the contents ofthe kit. The packaging material is constructed by well known methods,generally to provide a sterile, contaminant-free environment. Thepackaging material may have a label which indicates that thepolypeptides can be used for detecting antibody that specifically bindspolypeptides of the present invention. In addition, the packagingmaterial contains instructions indicating how the materials within thekit are employed to detect the antibody. As used herein, the term“package” refers to a container such as glass, plastic, paper, foil, andthe like, capable of holding within fixed limits the polypeptides, andother reagents, for instance a secondary antibody. Thus, for example, apackage can be a microtiter plate well to which microgram quantities ofpolypeptides have been affixed. A package can also contain a secondaryantibody. “Instructions for use” typically include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter, such as the relative amounts of reagent and sample to beadmixed, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions, and the like.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1 Preparation of Iron Regulated Proteins LaboratoryScale

Compositions derived from different strains of Staphylococcus aureusincluding novel proteins expressed under iron-restriction and/or otherdegrees of metal ion chelation were evaluated for efficacy against avirulent challenge in mice. The efficacy of the composition wasevaluated by collecting data on the following parameters (1) theefficacy of each composition to provide homologous and heterologousprotection against a live virulent challenge in mice, (2) the efficacyof each composition to reduce necrotic skin lesions, and (3) theefficacy of compositions derived from Staphylococcus grown in repleteand deplete iron conditions to provide protection.

The Staphylococcus aureus strains evaluated in this study originatedfrom three animal species; avian, human and bovine. The avian isolateSAAV1 was a field isolate originating from a flock of diseased turkeyshaving a high degree of osteomyelitis and synovitis. The bovine isolates(strain 1477 and strain 2176) were isolated from two differentcommercial dairy herds having a high incidence of clinical mastitis. Thehuman isolate was obtained from the ATCC (strain 19636), and originatedfrom a patient having clinical osteomyelitis.

Master seed stocks of each isolate were prepared by inoculating theappropriate isolate into 200 ml of Tryptic Soy Broth (TSB, DifcoLaboratories, Detroit, Mich.) containing 300 μM 2,2-dipyridyl(Sigma-Aldrich St. Louis, Mo.). The culture was grown while stirring at200 rpm for 6 hours at 37° C., and collected by centrifugation at10,000×g. The bacterial pellet was re-suspended into 100 ml TSB brothcontaining 20% glycerol, and sterilely dispensed into 2 ml cryogenicvials (1 ml per vial) and stored at −90° C. until use.

Each master seed stock was expanded into a working seed. One vial ofeach master seed isolate was inoculated into 200 ml of Tryptic Soy Broth(TSB, Difco Laboratories, Detroit, Mich.) containing 1000 μM2,2-dipyridyl (Sigma-Aldrich St. Louis, Mo.). The culture was grownwhile stirring at 200 rpm for 6 hours at 37° C., and collected bycentrifugation at 10,000×g. The bacterial pellet was resuspended into100 ml TSB broth containing 20% glycerol, and sterilely dispensed into 2ml cryogenic vials (1 ml per vial) and stored at −90° C. until use. Theworking seed was used for the production of compositions enriched withiron-regulated membrane proteins, including iron-regulated membraneproteins.

All strains were adapted to grow in highly iron-depleted media (i.e.,media containing very low levels of free iron). This was accomplished bysub-culturing the bacteria in TSB containing increasing concentrationsof 2,2-dipyridyl (from 300 to 1600 μM).

Proteins were prepared from bacteria as follows. The bacteria were grownfrom frozen working seed stocks by subculturing into 25 ml ofiron-deplete media (containing 1000 μM 2,2′-dyipyridyl) and iron-repletemedia, then incubated at 37° C. while shaking at 400 rpm. Following 12hours of incubation, 5 ml of each culture was transferred into 500 ml ofiron-deplete or iron-replete media pre-incubated at 37° C. Cultures wereincubated for 8 hours at 37° C. while shaking at 100 rpm, then cellswere pelleted by centrifugation at 10,000×g for 20 minutes. Bacterialpellets were resuspended in 100 ml of sterile physiological saline andcentrifuged at 10,000×g for 10 minutes. Pellets were then resuspended in45 ml of Tris-buffered saline, pH 7.2 (TBS; 25 mM Tris, 150 mM NaCl) andthe resulting bacterial suspensions were dispensed as 9-ml aliquots into5 individual tubes. One milliliter of TBS containing 50 units oflysostaphin (Sigma, St. Louis, Mo.) was added to each tube to give afinal volume of 5 units/ml. Following incubation at 37° C. for 30minutes while shaking at 200 rpm, 1 ml of TBS containing 0.1 mg oflysozyme (Sigma) was added to each tube. The bacterial suspensions werethen incubated for an additional 45 minutes while shaking at 200 rpm.Next, suspensions were centrifuged at 3050×g for 12 minutes at 4° C. topellet large cellular debris. The supernatants were collected byaspiration without disturbing the pellet. The supernatant was thencentrifuged at 39,000×g for 2.5 hours. The resulting pellets containingthe proteins were resusupended into 200 μL Tris buffer, pH 7.2, withoutsaline. The protein solution for each isolate were combined for a totalvolume of 1 ml and stored at −90° C.

The protein-enriched extracts derived from S. aureus weresize-fractionated on SDS-PAGE gels using a 4% stacking gel and 10%resolving gel. Samples for electrophoresis were prepared by combining 10μl of sample with 30 μl of SDS reducing sample buffer (62.5 mM Tris-HCLpH 6.8, 20% glycerol, 2% SDS, 5% β-mercaptoethanol) and boiled for 4minutes. Samples were electrophoresed at 18 mA constant current for 5hours at 4° C. using a Protein II xi cell power supply (BioRadLaboratories, Richmond, Calif., model 1000/500). The molecular weight ofeach individual protein as visually seen in the SDS-PAGE gel wasestimated using a GS-800 densitometer (BioRad) using a broad rangemolecular weight marker as a reference standard (BioRad).

The SDS-PAGE patterns of the proteins from each isolate when grown inthe presence of 1600 μM dipyridyl showed a very different proteinexpression pattern compared to the same strain when grown in thepresence of 300 μM dipyridyl. For instance, when grown in 300 μMdipyridyl isolate SAAV1 resulted in metal regulated proteins of 90 kDa,84 kDa, 72 kDa, 66 kDa, 36 kDa, 32 kDa, and 22 kDa, while growth in 1600μM dipyridyl resulted in metal regulated proteins of 87.73 kDa, 54.53kDa, 38.42 kDa, 37.37 kDa, 35.70 kDa, 34.91 kDa, and 33.0 kDa. Likewise,when grown in 300 μM dipyridyl isolate 19636 resulted in proteins of 42kDa and 36 kDa, while growth in 1600 μM dipyridyl resulted in metalregulated proteins of 87.73 kDa, 54.53 kDa, 38.42 kDa, 37.37 kDa, 35.70kDa, 34.91 kDa, and 33.0 kDa. All conditions, including growth iniron-replete media, resulted in the expression of the following proteinsthat were presumably not metal regulated: 150 kDa, 132 kDa, 120 kDa, 75kDa, 58 kDa, 50 kDa, 44 kDa 43 kDa 41 kDa, and 40 kDa.

Furthermore, growth of the different strains of S. aureus in 1600 μMdipyridyl resulted in similar protein expression patterns. Thecompositions enriched in iron-regulated membrane proteins from the avianisolate (SAAV1) included proteins with molecular weights of 87.73 kDa,54.53 kDa, 38.42 kDa, 37.37 kDa, 35.70 kDa, 34.91 kDa, and 33.0 kDa. Themolecular weights of the proteins from the ATCC isolate 19636 wereessentially identical to those from the avian isolate. Both bovineisolates, when grown with 1600 μM 2,2-dipyridyl, expressed similarbanding profiles as the avian and ATCC isolates for the majority of theproteins (87.73 kDa, 54.53 kDa, 37.7 kDa, 35.70 kDa, 34.91 kDa, and 33.0kDa). However, neither of the bovine isolates produced the 38.42 kDaprotein seen with the avian and ATCC isolates, and the bovine isolatesexpressed three proteins (80.46 kDa, 65.08 kDa, and 31.83 kDa) notobserved with the avian and ATCC strains (see FIG. 1 and Table 10). Allconditions resulted in the expression of the following proteins thatwere not metal regulated: 150 kDa, 132 kDa, 120 kDa, 75 kDa, 58 kDa, 50kDa, 44 kDa, 43 kDa, 41 kDa, and 40 kDa.

TABLE 10 Molecular weights of metal regulated polypeptides obtained fromStaphylococcus aureus isolates. Avian Human Bovine Bovine SAAV1 196361477 2176 87.73 87.73 87.73 87.73 — — 80.46 80.46 — — 65.08 65.08 54.5354.53 54.53 54.53 38.42 38.42 — — 37.37 37.37 37.37 37.37 35.70 35.7035.70 35.70 34.91 34.91 34.91 34.91 33.0  33.0  33.0  33.0  31.83 31.83

Interestingly, there was no difference in the protein profiles asexamined by SDS-PAGE between the clarified supernatant and the bacterialpellet after treating the bacteria with lysostaphin/lysozyme. Both theextracted bacterial pellet and the supernatant had exactly the sameprotein profiles as examined by SDS-PAGE. This same observation was alsoseen when disrupting the bacterial cells using an Avestin homogenizer at30,000 psi. The resultant bacterial pellet, after slow speedcentrifugation was identical in its protein profile as compared to theclarified supernatant after high speed centrifugation at 30,000×g for2.0 hours at 4° C.

Example 2 Preparation of the Immunizing Compositions Derived fromStaphylococcus aureus

The proteins from the human isolate ATCC 19636 and the bovine isolate1477, grown in iron-deplete conditions and prepared as described inExample 1, were used to formulate two vaccine compositions. The proteinsfrom the ATCC isolate had molecular weights of 87.73 kDa, 54.53 kDa,38.42 kDa, 37.37 kDa, 35.70 kDa, 34.91 kDa, and 33.0 kDa, while thebovine isolate expressed proteins having molecular weights 87.73 kDa,80.46 kDa, 65.08 kDa, 54.53 kDa, 37.37 kDa, 35.70 kDa, 34.91 kDa, 33.0kDa, and 31.83. Each composition also contained the following proteinsthat were not metal regulated: 150 kDa, 132 kDa, 120 kDa, 75 kDa, 58kDa, 50 kDa, 44 kDa, 43 kDa, 41 kDa, and 40 kDa. Stock vaccines wereprepared from the two strains by emulsifying each aqueous proteinsuspension (500 μg total protein/ml) into a commercial adjuvant(EMULSIGEN, MVP Laboratories, Ralston, Nebr.) using an IKA Ultra TurraxT-50 homogenizing vessel (IKA, Cincinnati, Ohio) to give a final dose of50 μg total protein in a 0.1 ml injectable volume with an adjuvantconcentration of 22.5% vol/vol. As a control vaccination, a proteincomposition was prepared from the bovine isolate 1477 grown underiron-replete conditions (TSB supplemented with 300 μM ferric chloride)as described in Example 1. A placebo vaccine was prepared bysubstituting physiological saline for the aqueous protein suspension inthe above protocol.

Example 3 Mouse Vaccination

Seventy (N=70) female CF-1 mice obtained from Harlan BreedingLaboratories (Indianapolis, Ind.) weighing 16-22 grams were equallydistributed into 7 groups (10 mice/group). Mice were housed inpolycarbonate mouse cages (Ancore Corporation, Bellmore, N.Y.). A singlecage was used for each treatment group and food and water was suppliedad libitum to all mice. All mice were vaccinated intraperitoneally with0.1 ml of the appropriate composition two times at 14 day intervals asfollows:

Group-1: Placebo-Vaccinated

Group-2: Vaccinated with ATCC 19636 proteins expressed underiron-restriction.

Group-3: Placebo-Vaccinated

Group-4: Vaccinated with Bovine 1477 proteins expressed underiron-restriction.

Group-5: Vaccinated with Bovine 1477 proteins expressed underiron-restriction.

Group-6: Vaccinated with ATCC 19636 proteins expressed underiron-restriction.

Group-7: Bovine 1477 FeCl₃-Vaccinated, where “Bovine 1477 FeCl₃” refersto proteins obtained from Bovine 1477 grown in TSB supplemented with 300μM ferric chloride.

Example 4 Preparation of Challenge Organism

The previously described Staphylococcus aureus strains ATCC 19636 andstrain 1477 were used as challenge organisms. Briefly, the isolates fromfrozen stocks (previously described) were streaked onto blood agarplates and incubated at 37° C. for 18 hours. A single colony of eachisolate was subcultured into 50 ml Tryptic Soy Broth (Difco) containing1600 μM 2,2′ dipyridyl. The cultures were incubated at 37° C. for 6hours while rotating at 200 rpm, then centrifuged at 10,000×g for 10minutes at 4° C. to pellet the bacteria. The bacterial pellets werewashed twice by centrifugation in TBS at 4° C. The final pellets wereresuspended in TBS to an optical density of 42% Transmittance (T) at 562nm in a volume of approximately 25 ml of TBS and used for challenge.Just prior to challenge, 1 ml of these bacterial suspensions wasserially diluted and plated on agar to enumerate the number ofcolony-forming units (CFU) per mouse dose.

Example 5 Challenge

Fourteen days after the second vaccination, mice in all groups (1-7)were subcutaneously challenged in the back of the neck with 0.1 ml ofthe appropriate organism. The seven groups of mice were challenged asfollows:

Group-1 (Placebo-Vaccinated): Challenged with ATCC 19636

Group-2 (Vaccinated with ATCC 19636 proteins expressed underiron-restriction): Challenged with ATCC 19636

Group-3 (Placebo-Vaccinated): Challenged with Bovine 1477

Group-4 (Vaccinated Bovine 1477 proteins expressed underiron-restriction): Challenged with Bovine 1477

Group-5 (Vaccinated Bovine 1477 proteins expressed underiron-restriction): Challenged with ATCC 19636

Group-6 (Vaccinated ATCC 19636 proteins expressed underiron-restriction): Challenged with Bovine 1477

Group-7 (Bovine 1477 FeCl₃-Vaccinated): Challenged with Bovine 1477

As determined by the enumeration protocol described in Example 4, theconcentration of S. aureus 19636 used for challenge was 1.35×10⁸ CFU permouse dose, and the concentration of S. aureus 1477 used for challengewas 1.65×10⁸ colony CFU per mouse dose. Morbidity, mortality and grosspathology were recorded daily for 7 days after challenge.

When comparing the mice challenged with the ATCC 19636 isolate, 70% ofthe placebo-vaccinated Group 1 mice died within 7 days of challenge(Table 11 and FIG. 2). This demonstrated that strain 19636 caused a highrate of mortality in mice at the dose level administered. In contrast tothe mice in Group 1, only 10% of the mice in Group 2 died within 7 dayspost-challenge. These results illustrated that the mice challenged withstrain 19636 were significantly protected by vaccination with the 19636composition (p=0.020, Fischer's Exact test). Furthermore, a Kaplan-Meieranalysis of the time-to-death data indicated that the vaccine affordedsignificant (p=0.0042, logrank test) protection against homologouschallenge (FIG. 3). In addition, only 20% of the mice in Group 5 diedwithin 7 days of challenge, indicating that the bovine 1477 compositionoffered significant protection against challenge with the ATCC 19636strain (p=0.015 logrank test for mortality). When the data was analyzedby a Kaplan-Meier survival curve and logrank test (FIG. 4), theprotection against mortality was determined to be significant (p=0.015logrank test for mortality), indicating that the vaccine compositionderived from strain 1477 provided heterologous protection againstchallenge with strain 19636.

TABLE 11 Mortality of Vaccinated and Non-Vaccinated Mice FollowingChallenge with Staphylococcus aureus (human ATCC isolate 19636 andbovine isolate 1477). Percent Groups # Mice # Dead mortality (%)Group-1* (Placebo, ATCC 19636 10 7/10 70 Chlg) Group-2* (ATCC 19636, 101/10 10 Homologous Chlg) Group-3* (Placebo, Bovine 1477 10 2/10 20 Chlg)Group-4* (Bovine 1477, 10 1/10 10 Homologous Chlg) Group-5* (Bovine1477, 10 2/10 20 Heterologous Chlg) Group-6* (ATCC 19636, 10 0/10 0Heterologous Chlg) Group-7* (Bovine 1477 FeCl₃, 10 2/10 20 Bovine 1477Chlg) *Group-1, (Placebo-Vaccinated/Challenged with ATCC 19636) *Group-2(Vaccinated with ATCC 19636 proteins expressed underiron-restriction/Challenged with ATCC 19636) *Group-3(Placebo-Vaccinated/Challenged with Bovine 1477) *Group-4 (Vaccinatedwith Bovine 1477 proteins expressed under iron-restriction/Challengedwith Bovine 1477) *Group-5 (Vaccinated with Bovine 1477 proteinsexpressed under iron-restriction/Challenged with ATCC 19636) *Group-6(Vaccinated with ATCC 19636 proteins expressed underiron-restriction/Challenged with Bovine 1477) *Group-7 (Bovine 1477FeCl₃ - Vaccinated/Challenged with Bovine 1477)

When comparing the mice challenged with the bovine 1477 isolate, only20% of the mice in the placebo-vaccinated group (Group 3) died within 7days of challenge. However, challenge with the bovine 1477 isolateelicited the development of necrotic skin lesions on 6 (75%) of thesurviving mice of Group 3. These lesions were measured and the averagesize of the lesions on the surviving mice was 18.5 mm (Table 12). Incontrast, 20% of the Group 4 mice died within 7 days of challenge, butonly three (38%) of the surviving mice developed lesions (averagediameter, 2.7 mm). These results indicate that the bovine 1477composition offered significant homologous protection againstdevelopment of lesions in the mice challenged with the bovine strain1477 (p=0.009, Student's t-test). In addition, vaccination with the ATCC19636 composition protected against challenge with strain 1477, since nomice died in Group 6 and only three (30%) of the mice developed skinlesions (average diameter, 3.7 mm). Taken together, the reducedmortality and/or lesion development in the mice in Groups 5 and 6demonstrate the significant cross-protective nature of the compositionsderived from strains 19636 and 1477 (p=0.012, Student's t-test based onlesion size). In demonstration of the efficacy of the composition ascompared to the non-iron regulated proteins, 20% of the mice in Group 7died and 4 of the survivors developed skin lesions (average diameter,15.8 mm). The mice of Group 7 demonstrated some degree of protection byvaccination with the proteins of the 1477 isolate since fewer micedeveloped lesions compared to the placebo-vaccinated Group 3. However,the skin lesions observed on the mice in group 7 were more frequent andof a larger diameter than the lesions on the mice of Group 4, indicatingthat, relative to proteins isolated from cells grown under iron-repleteconditions, the proteins isolated from bacteria grown under ironrestriction offered superior protection against an identical challenge.

TABLE 12 The Induction of Necrotic Lesions in Mice Seven DaysPost-Challenge with Staphylococcus aureus (ATCC Isolate 19636 and/orBovine Isolate 1477) Group-1 Group-2 Group-3 Group-4 Group-5 Group-6Group-7 Lesion diameter (millimeter) per mouse No lesion No lesion 26 55 5 25 No lesion No lesion 25 2 No lesion 5 25 No lesion No lesion 24 1No lesion 1 10 Dead No lesion 24 No lesion No lesion No lesion 3 Dead Nolesion 7 No lesion No lesion No lesion No lesion Dead No lesion 5 Nolesion No lesion No lesion No lesion Dead No lesion No lesion No lesionNo lesion No lesion No lesion Dead No lesion No lesion No lesion Nolesion No lesion No lesion Dead No lesion Dead No lesion Dead No lesionDead Dead Dead Dead Dead Dead No lesion Dead Average lesion diameter(mm) among surviving mice 0 0 18.5 2.7 5 3.7 15.8 *Group-1,(Placebo-Vaccinated/Challenged ATCC 19636) *Group-2 (Vaccinated withATCC 19636 proteins expressed under iron-restriction/Challenged ATCC19636) *Group-3 (Placebo-Vaccinated/Challenged Bovine 1477) *Group-4(Vaccinated with Bovine 1477 proteins expressed underiron-restriction/Challenged Bovine 1477) *Group-5 (Vaccinated withBovine 1477 proteins expressed under iron-restriction/Challenged ATCC19636) *Group-6 (Vaccinated with ATCC 19636 proteins expressed underiron-restriction/Challenged Bovine 1477) *Group-7 (Bovine 1477 FeCl₃Vaccinated/Challenged Bovine 1477)

The cross-protective nature of the proteins observed in the mousechallenge study is supported by the similar molecular weights of theproteins from the S. aureus strains described in Example 1 (FIG. 1).Although there were noticeable differences in the SDS-PAGE profile ofthe proteins from the bovine-derived isolates, specifically the absenceof a 38.4 kDa protein and the presence of 3 additional proteins, theproteins from both strains 1477 and ATCC 19636 elicited heterologousprotection. These results indicate that the similar proteins betweenstrains 19636 and 1477 are likely responsible for the cross-protectionobserved in Groups 5 and 6. By contrast, the protein profiles fromstrain 1477 grown under iron-deplete and iron-replete conditions areobservably different. Those proteins isolated under iron-depletedconditions are more protective when compared to the proteins isolatedunder iron-replete conditions, demonstrated by the reduction in lesiondevelopment among the mice of Group 4 compared to the mice of Group 7.

Example 6

In mammals, it has been shown that the response to tissue injury orbacterial infection results in an acute inflammatory response. Thisresponse increases capillary permeability and phagocytic infiltrationresulting in the clinical signs recognized as inflammation; swelling,fever, pain and redness; if left uncontrolled, this may lead to death.The activation of humoral factors and the release of cytokines mediatesystemic events collectively known as the acute phase protein responsewhich results in a cascade of physiological and biochemical events. Theduration of this response is directly related to the severity of theinjury and magnitude of the systemic infection. It has beenwell-documented that during bacterial sepsis, major surgery, burns andother bodily trauma there is an alteration in the concentration of anumber of metal ions in serum such as, iron, copper, and zinc. Forinstance, during the acute phase of an infection there is a decrease inplasma levels of iron and zinc and an increase in copper. The alterationof these trace metal ions in serum may directly affect the severity orprogression of any bacterial infection.

In this study we examined the expression of proteins of Staphylococcusaureus under various conditions of metal ion restriction in order tomimic the expression of novel proteins that may be expressed duringsystemic invasion. The Staphylococcus aureus strains evaluated in thisstudy originated from clinical samples of three different species ofanimal; avian (strain SAAV1), human (strain 19636), and bovine (strains1477 and 2176). Briefly, cultures of each isolate were prepared frommaster seed stocks in 200 ml of Tryptic Soy Broth (TSB). Each culturewas grown while stirring at 200 rpm for 6 hours at 37° C. Ten ml of eachculture were transferred into 500 ml of deplete TSB containing one offour metal ion chelators; 2,2-dipyridyl (Dp), 2-pyridylmethyl-ethylenediamine (TPEN), catechin, and naringenin (all obtained from Sigma, St.Louis, Mo.). In addition each culture was also grown in cation-repletemedia containing ferric chloride, zinc chloride and/or copper chlorideprepared at 300 μM concentrations. The metal ion chelators were used atthe following concentration; 2,2-dipyridyl (800 μM), catechin andnaringenin were used at 300 μM, and 2-pyridylmethyl-ethylene diamine wasused at a concentration of 100 μM. Cultures were grown with eachchelator for 8 hours, at which point the culture was subcultured asecond time for an additional 12 hours. Each culture was subcultured forthree consecutive passes at 12-hour intervals. At the end of the thirdpass, each culture was harvested by centrifugation at 10,000×g for 20minutes. Each culture was washed twice by centrifugation at 10,000×g andresuspended in 20 ml Tris-buffered saline, pH 7.2 at 4° C.

Each bacterial pellet was resuspended in 45 ml of Tris-buffered saline,pH 7.2 (25 mM Tris and 150 mM NaCl) and the resulting bacterialsuspensions were dispensed as 9-ml aliquots into 5 individual tubes,twenty tubes total. One milliliter of TBS containing 50 units oflysostaphin (Sigma, St. Louis, Mo.) was added to each tube to give afinal concentration of 5 units/ml. Following incubation at 37° C. for 30minutes while shaking at 200 rpm, 1 ml of TBS containing 0.1 mg oflysozyme (Sigma) was added to each tube. The bacterial suspensions werethen incubated for an additional 45 minutes while shaking at 200 rpm.Next, suspensions were centrifuged at 3050×g for 12 minutes at 4° C. topellet large cellular debris. The supernatants were collected byaspiration without disturbing the pellet. The supernatant was thencentrifuged at 39,000×g for 2.5 hours. The resulting pellets, enrichedfor metal-regulated membrane proteins, were resuspended in 200 μL Trisbuffer, pH 7.2. The protein solutions for each isolate were combined fora total volume of 1 ml and stored at −90° C.

The proteins obtained from the SAAV1, 19636, 1477 and 2176 S. aureusisolates grown under iron, zinc and copper deplete conditions includedmetal-regulated polypeptides.

Cell extracts, derived from each isolate were size-fractionated onSDS-PAGE gels using a 4% stacking gel and 10% resolving gel. Samples forelectrophoresis were prepared by combining 10 μl of sample with 30 μl ofSDS reducing sample buffer (62.5 mM Tris-HCL ph 6.8, 20% glycerol, 2%SDS, 5% beta-mercaptoethanol) boiled for 4 minutes. Samples wereelectrophoresed at 18 mA of constant current for 5 hours at 4° C. usinga Protein II xi cell power supply (BioRad Laboratories, Richmond,Calif., model 1000/500).

The SDS-PAGE patterns of the proteins grown under zinc and/or copperchelation showed unique banding patterns in all isolates that weredifferent when compared to the same isolates grown underiron-restriction in the presence of 2,2′-dyipyridyl. For example, whenthe 19636 isolate was grown under iron-restriction or in the presence ofthe chelator 2,2′-dyipyridyl, unique iron-regulated proteins wereexpressed at the 87.73 kDa, 54.53 kDa, 38.42 kDa, 37.37 kDa, 35.70 kDa,34.91 kDa and 33.0 kDa regions. These proteins were downregulated whenthe isolate was grown in the presence of ferric chloride. However, whenthe same isolate was grown in the presence of the zinc and or copperchelator, a novel subsets of proteins was expressed relative to theproteins expressed under iron-restriction; the new proteins havingmolecular weights of approximately 115 kDa, 88 kDa, 80 kDa, 71 kDa, 69kDa, 35 kDa, 30 kDa, 29, kDa and 27 kDa. In addition, an 87.73 kDaprotein was expressed under conditions of iron restriction orcopper-restriction but not when cultures were zinc-restricted. Theproteins expressed under iron-restriction appeared to be downregulatedwhen growth was under either zinc-restriction and/or copper-restriction,but not completely shut off as seen when the isolate was grown in ferricchloride.

It appears that there are novel proteins expressed when the organism isgrown under copper-restriction and/or zinc-restriction that are notexpressed when the same isolate is grown under iron-restrictedconditions. Since transitional metals are used by organisms to buildenzymes that catalyze various biochemical reactions, the metal ions mayplay a vital role in microbial survival during a systemic infection. Itis perhaps for this reason that during sepsis there is a transientdecrease in the availability of these transitional metals, making themunavailable for growth of the organism. These novel proteins could verywell enhance the protective efficacy of the existing composition grownunder iron-restriction because they may also be expressed by thebacteria under the metal ion restriction experienced during systemicinvasion.

Example 7 Compositions of the Present Invention can Also be ProducedUnder Large Scale Commercial Conditions Fermentation

A cryogenic vial of the working seed (2 ml at 10⁹ CFU/ml) as describedin Example 1 was used to inoculate 500 ml of Tryptic Soy Broth (TSB)without dextrose (Difco) pre-warmed to 37° C. containing 0.125g/12,2-dipyridyl (Sigma), 2.7 grams BiTek yeast extract (Difco) andglycerol (3% vol/vol). The culture was incubated at 37° C. for 12 hourswhile stirring at 200 rpm at which time it was used to inoculate 2liters of the above media and allowed to grow for an additional 4 hoursat 37° C. This culture was used to inoculate a 20-liter Virtis bench-topfermentor, (Virtis, Gardiner, N.Y.) charged with 13 liters of theabove-described media. The pH was held constant between 6.9 and 7.1 byautomatic titration with 50% NaOH and 10% HCL. The stirring speed wasadjusted at 400 rev/minute, and the culture aerated with 11 litersair/minute at 37° C. Foaming was controlled automatically by theaddition of 11 ml defoamer (Mazu DF 204 Chem/Serv, Minneapolis, Minn.).The culture was allowed to grow continuously at these conditions for 4hours at which time was sterilely pumped into a 150-liter fermentor (W.B. Moore, Easton, Pa.). The fermentor was charged with 120 literstryptic soy broth without dextrose (3,600.0 grams), BiTek yeast extract(600 grams), glycerol (3,600 ml), 2,2-dypyrdyl (3.0 grams) and Mazu DF204 defoamer (60 ml). The parameters of the fermentation were asfollows: dissolved oxygen (DO) was maintained at 30%+/−10% by increasingagitation to 220 rev/minute sparged with 60 liters of air/minute and 10pounds per square inch (psi) back pressure. The pH was held constantbetween 6.9 and 7.1 by automatic titration with 50% NaOH and 10% HCL andthe temperature maintained at 37° C. At hour 4.5 (OD₅₄₀ 8-9) of thefermentation the culture was transferred to a 1,500 liter New BrunswickScientific fermentor IF-15000 charged with 1200 liters tryptic soy brothwithout dextrose (36,000 grams), BiTek yeast extract (6,000 grams),glycerol (36,000 ml), 2,2-dypyrdyl (30.0 grams) and Mazu DF 204 defoamer(600 ml). The parameters of the fermentation were as follows: dissolvedoxygen (DO) was maintained at 60%+/−10% with supplemental oxygen byincreasing agitation to 300 rev/minute sparged with 300 to 1100 litersof air/minute and 5 pounds per square inch (psi) back pressure. Asfermentation progressed supplemental oxygen was added from 0-90liters/minute to assist in the control of dissolved oxygen. The pH washeld constant between 6.9 and 7.4 by automatic titration with 50% NaOHand 10% HCL and the temperature was maintained at 37° C.

At approximately 5 hours post inoculation of the large fermentor theculture was supplemented with additional nutrients by feeding 70 litersof media containing 18,000 grams TSB without dextrose, 3,000 grams yeastextract 30.0 grams 2,2-dipyridyl and 18,000 ml of glycerol. The rate offeed was adjusted to approximately 28 liters/hour while increasingagitation. At the end of the feed the fermentation was allowed tocontinue for an additional 4 hours at which point the fermentation wasterminated by lowing the temperature of the fermentor to 18° C. (OD₅₄₀3540 at a 1:100 dilution).

Harvest

The bacterial fermentation was concentrated and washed using a PallFiltron Tangential Flow Maxiset-25 (Pall Filtron Corporation, Northboro,Mass.) equipped with three 30 ft² Alpha 300-K open channel filters,catalog No. AS300C5, (Pall Filtron) connected to a Waukesha Model U-60feed pump (Waukesha Chemy-Burrell, Delevan, Wis.) The original culturevolume of 1250 liters was reduced to 50 liters (2.5 liters/minute) usinga filter inlet pressure of 30 psi and a retentate pressure of 5-6 psi.The bacterial retentate was adjusted back up to 150 liters usingTris-buffered Saline pH 8.5 and then concentrated again to 50 liters tohelp remove any contaminating exogenous proteins, such as exoproteins toinclude secreted toxins and proteases. The elevated pH of thetris-buffered saline helps prevent much of the proteolytic degradationthat can occur during storage of the whole cell suspension. Proteaseinhibitors may be used instead of, or in addition to, an elevated pH.The retentate was mixed thoroughly while in the 200-liter tank using abottom mount magnetically driven mixer. The retentate was sterilelydispensed (3.5 liters) into sterile 4 liter Nalgene containers No. 2122and placed into a −20° C. freezer for storage as a breaking point in themanufacture, or could be further processed. The pellet mass wascalculated by centrifuging 30 ml samples of the fermented culture andfinal harvest. Briefly, pre-weighted 50 ml Nalgene conical tubes werecentrifuged at 39,000×g for 90 minutes in a Beckman J2-21 centrifugeusing a JA-21 rotor (Beckman Instruments, Palo Alto Calif.). At the endof the run, the supernate was poured off and the tubes were weighedagain. The pellet mass was calculated for each stage. The fermentationprocess yielded a wet pellet mass of approximately 60 kilograms.

Disruption

Eighty kilograms of bacterial cell slurry in Tris-buffered Saline pH 8.5was aseptically transferred into a steam in place 1000 liter jacketedprocess tank (Lee, Model 259LU) with a top mounted mixer (Eastern, ModelTME-1/2, EMI Incorporated, Clinton, Conn.) containing 900 liters TBS pH8.5. The bulk bacterial suspension was chilled to 4° C. with continuousmixing for 18 hours at 200 rpm at which time was disrupted byhomogenization. Briefly, the 1000 liter tank containing the bacterialsuspension was connected to a model C-500-B Avestin Homogenizer,(Avestin Inc, Ottawa Canada). A second 1000 liter jacketed process tank(empty) was connected to the homogenizer such that the fluid in theprocess tank could be passed through the homogenizer, into the emptytank and back again, allowing for multiple homogenizing passes whilestill maintaining a closed system. The temperature during homogenizationwas kept at 4° C. At the start of the first pass, fluid was circulatedat 70 psi via a Waukesha model 10DO pump (Waukesha) through thehomogenizer (500 gallons/hour), while the homogenizer pressure wasadjusted to 30,000 psi. Prior to the first pass, two pre-homogenizingsamples were withdrawn from the homogenizer to establish a baseline fordetermining the degree of disruption and monitoring of pH. The degree ofdisruption was monitored by transmittance (% T at 540 nm at 1:100dilution) compared to the non-homogenized sample. The number of passesthrough the homogenizer was standardized to give a final percenttransmittance between 78-91% T at a 1:100 dilution preferably between86-91%. After homogenization, the tank was removed from the homogenizerand put onto a chiller loop at 4° C. and mixed at 240 rpm.

Protein Harvest

The disrupted bacterial suspension containing the iron-regulatedproteins as illustrated in FIG. 1 were collected by centrifugation usingT-1 Sharples, (Alfa Laval Seperations, Warminster, Pa.). Briefly, the1000 liter jacketed process tank containing the disrupted bacterialhomogenate was fed into 12 Sharples with a feed rate of 250 ml/minute at17 psi at a centrifugal force of 60,000×g. The effluent was collectedinto a second 1000 liter jacketed process tank through a closed sterileloop allowing for multiple passes through the centrifuges whilemaintaining a closed system. The temperature during centrifugation waskept at 4° C. The homogentae was passed 8 times across the centrifuges.Approximately 50% of the protein was collected after the second pass, atwhich point, the homogenate fluid was concentrated to ⅓ of its originalvolume, which shortened the process time for the next 6 passes. Thehomogenate tank was aseptically disconnected from the centrifuges andconnected to a Millipore Pellicon Tangential Flow Filter assembly(Millipore Corporation, Bedford, Mass.), equipped with a 25 ft²screen-channel series Alpha 30K Centrasette filter (Pall Filtron)connected to a Waukesha Model U30 feed pump for concentration. Afterconcentration, centrifugation was continued until the process wascompleted. Protein was collected after each pass. The protein wascollected, resuspended and dispensed in 50 liters Tris-buffered salinepH 8.5 containing 0.15% formulin (Sigma) as preservative.

Diafiltration

The protein suspension was washed by diafiltration at 4° C. to removeany exogenous proteins (proteases, toxins, cytoplasmic and metabolicenzymes etc). Briefly, the 50 liters of protein was sterilelytransferred into a 200 liter process tank containing 150 liters sterileTris-buffer saline, pH 8.5 equipped with a bottom mount Dayton mixer,Model 2Z846 (Dayton Electric, Chicago, Ill.) rotating at 125 rev/minute.The process tank was sterilely connected to a Millipore PelliconTangential Flow Filter assembly (Millipore Corporation), equipped with a25 ft² screen-channel series Alpha 30K Centrasette filter (Pall Filtron)connected to a Waukesha Model U30 feed pump. The 200 liter proteinsolution was concentrated by filtration to a target volume 50 liters atwhich point 150 liters of sterile saline was added. The proteinsuspension was then concentrated to approximately 50 liters. The proteinconcentrate was stored in a 50 liter jacketed process tank equipped witha top mounted mixer and stored at 4° C.

It is interesting to note that the composition derived from the largescale process using homogenization as a means of disruption generatedidentical banding profiles as examined by SDS-PAGE as compared to thesmaller scale process described in Example 1. These results show thatlysostaphin could be replaced as the bacterial lysis agent using theAvestin homogenizer C500-B. This discovery allows for the low costgeneration of large volumes of iron-regulated proteins fromstaphlylococci.

Example 8 Hyper-Immunization of Mice and Preparation of PolyclonalAntibody

Passive immunization with purified antibody isolated from micevaccinated with proteins derived from S. aureus strains 19636 grownunder iron-limiting conditions was protective against a homologous andheterologous S. aureus challenge. Fifteen adult CD1 mice were vaccinatedas described in Example 3 with the protein composition derived from S.aureus strain ATCC19636 grown under iron-deplete conditions as describedin Examples 1 and 2. Mice were vaccinated intraperitoneally 3 times at 7day intervals with 50 μg of protein composition at each vaccination.Seven days after the third immunization, mice were bled completely bycardiac puncture. Serum was pooled and antibody purified using standardammonium sulfate precipitation. Exogenous serum proteins were removedfirst prior to antibody precipitation by adding 0.5 volumes of saturatedammonium sulfate pH 7.2. The solution was stirred at 100 rpm for 24hours at 4° C. The solution was again centrifuged at 3000×g for 30minutes. The supernatant was collected and precipitated again by addingenough saturated ammonium sulfate to bring the final concentration to55% saturation. The solution was stirred at 100 rpm for 24 hours at 4°C. The precipitate was centrifuged at 3000×g for 30 minutes. The finalpellet from each sample was resuspended into 2 ml PBS pH 7.2. Theprecipitated antibodies were then dialyzed using a 50,000 molecular cutoff dialysis tubing (Pierce, Rockford Ill.) for 30 hours against three 1liter changes of phosphate-buffered saline to remove ammonium sulfate.The first two liter changes were preserved with 0.02% sodium azide. Thefinal 1 liter buffer change contained no preservative. The dialysate wascollected and centrifuged again to remove any remaining debris at 3000×gfor 30 minutes. The antibody solution was stored at 4° C. for less then48 hours prior to use. Each sample was plated on blood agar to verifysterility prior to infusion.

Example 9 Passive Immunization and Challenge

In order to evaluate the protective effect of infused antibody raisedagainst S. aureus proteins expressed during iron-limitation, two groupsof 15 mice each were infused intraperitoneally with either the purifiedantibody preparation (Group 1) or physiological saline (Group 2) in a200 mL infusion. An additional two groups of 15 mice each were infusedsubcutaneously with either the purified antibody preparation (Group 3)or physiological saline (Group 4). After 60 minutes, the 2 groups of 15mice receiving an intraperitoneal infusion were challengedintraperitoneally with 1.3×10⁸ cfu of S. aureus strain 19636. Similarly,the 2 groups of 15 mice receiving a subcutaneous infusion werechallenged subcutaneously with 1.3×10⁸ cfu of S. aureus strain 1477 totest for cross-protection against challenge by a different S. aureusstrain. Mortality and/or lesion size was recorded for 5 days and thelivers of all mice were removed post-mortem, homogenized and plated todetermine the number of S. aureus present as a measure of systemicinfection. The Kaplan-Meier survival curves (FIGS. 5 and 6) show theprotective effect afforded by the infusion of antibodies from micevaccinated with the S. aureus proteins expressed during ironrestriction. Although the difference between the infused and controlgroups for the ATCC 19636-challenge groups was not significant (p=0.076,log-rank test), the liver of the single mouse that died within theantibody-infused group at Day 1 was cultured on blood agar to determinethe absence and/or presence of the challenge organism (S. aureus). Theculture derived from this mouse was negative for Staphylococcus andshowed no growth on the blood agar plate or culture medium. In contrast,the livers of the mice that died within the placebo group, were allpositive for the presence of Staphylococcus, in fact, pure cultures wereobtained on each blood agar plate derived from the livers of these mice.While the liver data do not preclude the possibility that the mouse thatdied within the antibody-infused group died of S. aureus infection, theinfection was not systemic, as it was in the placebo group, and themouse may have died for other reasons. Censoring of thisantibody-infused mouse death results in a significant difference betweenantibody-infused and placebo treatments (p=0.015, log-rank test). Thedata for the cross-challenge, where mice were infused with antibodygenerated after vaccination with ATCC 19636-derived proteins andchallenged by S. aureus strain 1477, also showed a protective trend.Between 7 and 14 days post challenge, all mice in the infused andnon-infused groups began to develop necrotic skin lesions. However,gross examination of mice clearly revealed a visible delay in theformation of an observable lesion as well as the severity of the lesionbetween the groups. Infused mice developed lesions more slowly ascompared to non-infused control mice which developed lesion faster theninfused mice and at a greater degree of severity. The infused micehealed faster then non-infused mice. This was clearly evident between 21and 35 days post challenge. Gross examination of mice at 35 days postchallenge showed that non-infused mice were severely disfigured andrevealed a greater degree of scarring. In fact, many of these mice lostnormal posture, in that they appeared twisted in appearance, in contrastto infused mice that did not develop nearly the extensive scar tissueand/or disfigurement as illustrated by the twisted appearance that thenon-infused mice developed. Overall, these data suggest thatinterperitoneal infusion of antibodies raised against S. aureusiron-induced proteins can both protect against and limit the severity ofS. aureus infection.

Example 10 Evaluation of a Vaccine Composition Derived fromStaphylococcus aureus in a Chronically Infected Dairy Herd

A commercial Dairy herd having a history of chronically high somaticcell counts attributable to Staphylococcus aureus was chosen for theevaluation of a vaccine composition as described in Example 1. Thecriterion for establishing vaccine efficacy of this experimental studywas: 1) decreased incidence of clinical mastitis caused byStaphylococcus aureus among vaccinates compared to non-vaccinatedcontrols, 2) improvement (i.e., a decrease) in somatic cell count amongvaccinates compared to controls and 3) decrease in culture positiveisolation rates of S. aureus between vaccinated and non-vaccinatedcontrols. Blood will be taken at the time of the first vaccination (day0) and again at 3 and 6 weeks post initial immunization. Injection sitereactions or systemic reactions following vaccinations were monitoredthroughout the study. In addition, bulk tank milk samples were culturedand quantitatively enumerated to determine if there was a decrease inthe number of CFU of Staphylococcus aureus cultured after vaccination.

Three of the Staphylococcus isolates derived from the chronicallyinfected lactating cows within the herd were grown under conditions ofiron-restriction and non-iron restricted conditions as described inExample 1. The three isolates were designated TTX101, TTX102, andTTX103. Extracted samples were examined by SDS-PAGE to compare bandingprofiles between isolates. Identical banding profiles were observedamong isolates examined; the compositions made from each isolateincluded proteins having molecular weights of 87.73 kDa, 80.46 kDa,65.08 kDa, 54.53 kDa, 37.37 kDa, 35.70 kDa, 34.91 kDa, 33.0 kDa and31.83 kDa. These proteins are the same molecular weights as previouslydescribed in Table 10. In addition, when comparing the isolatesidentical banding profiles were seen with those proteins that wereexpressed in all conditions that were not regulated by iron: 150 kDa,132 kDa, 120 kDa, 75 kDa, 58 kDa, 50 kDa, 44 kDa, 43 kDa, 41 kDa, and 40kDa. These results were consistent with previous observations. Oneisolate designated as TTX101 was chosen as the isolate to manufacture acomposition to be used in this study.

Example 11 Vaccine Preparation of Staphylococcus aureus (TTX101)

A composition was prepared as described in Example 1 using the isolateTTX101. The composition included proteins expressed under iron depleteconditions having molecular weights of 87.73 kDa, 80.46 kDa, 65.08 kDa,54.53 kDa, 37.37 kDa, 35.70 kDa, 34.91 kDa, 33.0 kDa, and 31.83 kDa aswell as non-metal regulated proteins having molecular weights of 150kDa, 132 kDa, 120 kDa, 75 kDa, 58 kDa, 50 kDa, 44 kDa 43 kDa 41 kDa, and40 kDa. The immunizing composition derived from strain TTX101 was usedto prepare the experimental vaccine by emulsifying the extracted proteinsuspension (400 μg total protein per milliliter) into a commercialadjuvant (EMULSIGEN, MVP Laboratories, Ralston Nebr.) using an IKA UltraTurrax T-50 homogenizing vessel (IKA, Cincinnati, Ohio) to give a finaldose of 800 μg total protein in a 2.0 ml injectable volume with anadjuvant concentration of 22.5% vol/vol. The vaccine was administeredsubcutaneously 2 times at 21 day intervals.

Example 12 Experimental Design and Herd Vaccination

Eighteen days before the first vaccination all lactating cows enrolledin the study (N=80) were tested for S. aureus by standardized aerobicbacteriological culture methods by culturing individual milk samplesderived from each lactating cow. In addition, the Somatic Cell Counts(SCC) were enumerated by the Dairy Herd Improvement Association usingstandard methods. Fourteen of the 80 cows were clinically diagnosed withmastitis and were culture positive for S. aureus. The remaining cows(N=66) tested negative for S. aureus. The eighty cows were equallydivided into two groups designated as group-1, vaccinated (N=40) andgroup-2, non-vaccinated (N=40). The fourteen clinically diagnosedStaphylococcus positive cows were equally distributed between bothgroups so that each study group contained 7 cows with clinical mastitis.The average SCC between groups prior to the first vaccination was203,219 in the non-vaccinated controls compared to 240,443 in vaccinates(not statistically different p=0.7).

Eighteen days after the first sampling all cows in group 1 werevaccinated subcutaneously in the upper right shoulder with 2 ml ofvaccine as described in Example 11. Ten days after the first vaccinationmilk samples were taken at this time period by the DHIA for theenumeration of somatic cells from each individual cow. Milk samples werenot bacteriologically tested at this time period for determining thepresence of Staphylococcus. The difference in the SCC between groups atthis time period was 125,241 (vaccinates) compared to 196,297(controls). This was a 36% difference in the number of somatic cellsbetween vaccinates as compared to non-vaccinated controls. Thedifference in the SCC between the controls and vaccinates at thissampling period was not statistically different (p=0.5). The lack ofstatistical difference in the SCC between groups at both samplingperiods was due to the large variation in individual SCC between cows.The injection site of each vaccinated cow was also examined at this sametime period. None of the cows examined showed any adverse tissuereaction at the site of injection by physical examination. In addition,there was no measurable loss in milk production due to vaccination.

Twenty one days after the first vaccination all cows in group-1(vaccinates) were given their second vaccination or booster. During thetime period between first and second vaccination, cows in both groups(vaccinates and controls) developed teat damage due to a dramatic dropin the environmental temperature resulting in the formation of lesionsat the end of the teat, resulting in the development of infected teatsand potentially increasing the isolation of Staphylococcus duringsampling, which was observed at the third sampling period. Twenty threedays after the second vaccination milk samples were taken by the DHIAfor the enumeration of Somatic Cells from each individual cow. Milksamples were also bacteriologically tested for the presence ofStaphylococcus aureus. There was a dramatic increase in isolation rateof S. aureus at this time period in the cows that tested negative at thefirst sampling period. In the non-vaccinated controls 42.9% of thesecows now tested positive for S. aureus, in contrast to the vaccinates,which only showed and increase of 35.5%. This was a 7.4% differencebetween vaccinates as compared to the non vaccinated controls. It'sdifficult to say that the improvement in the isolation rate of S. aureusin the vaccinated group was due to the effect of the vaccine alone. Onecannot overlook the difficulty in obtaining clean milk samples from cowsthat had teat damage which could increase the potential contamination ofthe milk by S. aureus when obtaining the sample. Nevertheless, there wasa significant difference in the average SCC between vaccinates comparedto controls. The average SCC of the vaccinated group was 222,679compared to 404,278 somatic cells as measured in the control group. Thiswas a 44.9% difference between vaccinates when compared to the nonvaccinated controls. It's interesting to speculate that the differenceseen in the SCC between these groups also coincides with the differencein the isolation rate of S. aureus between groups. However, due to thelarge variation in SCC between individual animals and the small samplesize of the experimental trial in the number of animals the differencewas not statistically different (p=0.28).

At this same time period the injection site of each vaccinated cow wasexamined for any adverse tissue reaction that may have been caused bythe vaccine composition. None of the cows examined showed any adversereaction at the site of injection by physical examination. The vaccinecompositions appeared to be highly tissue compatible and caused nomeasurable loss in milk production after each vaccination.

Monitoring of the cows is continued by measuring SCC and milk samplesfor the presence or absence of Staphylococcus aureus. Some of the cowsof each group are vaccinated a third time at 42 days after the secondvaccination. There appears to be a difference favoring the use of thevaccine composition for decreasing somatic cell counts and controllinginfection caused by Staphylococcus aureus. Further monitoring includesserology based on antibody titers to the vaccine composition, changes inmilk production in vaccinated cows due the improvement in health, anddecreased SCC of vaccinated animals compared to non-vaccinated cohorts.In addition, other experiments are conducted to investigate theprotective index of the vaccine based on dose response followingchallenge with a virulent S. aureus.

Example 13

Since the molecular weights of the proteins among the different S.aureus strains have been demonstrated to be similar and sinceheterologous protection was observed in the mouse challenge study, wesought to determine if the proteins sharing similar molecular weights inFIG. 1 were similar proteins. The technique chosen to characterize theproteins was matrix-assisted laser desorption/ionization massspectrometry (MALDI-MS). A portion of the composition was resolved usingSDS-PAGE as described in Example 1, and the gel was stained withCoomassie Brilliant blue to visualize the proteins.

Materials and Methods

Excision and washing. The gel was washed for 10 minutes with watertwice. Each protein band of interest was excised by cutting as close tothe protein band as possible to reduce the amount of gel present in thesample.

Each gel slice was cut into 1×1 mm cubes and placed in 1.5 ml tube. Thegel pieces were washed with water for 15 minutes. All the solventvolumes used in the wash steps were approximately equal to twice thevolume of the gel slice. The gel slice was next washed withwater/acetonitrile (1:1) for 15 minutes. When the proteins had beenstained with silver, the water/acetonitrile mixture was removed, the gelpieces dried in a SpeedVac (ThermoSavant, Holbrook, N.Y.) and thenreduced and alkylated as described below. When the gel pieces were notsilver-stained, the water/acetonitrile mixture was removed, andacetonitrile was added to cover until the gel pieces turned a stickywhite, at which time the acetonitrile was removed. The gel pieces wererehydrated in 100 mM NH₄HCO₃, and after 5 minutes, a volume ofacetonitrile equal to twice the volume of the gel pieces was added. Thiswas incubated for 15 minutes, the liquid removed, and the gel piecesdried in a SpeedVac.

Reduction & alkylation. The dried gel pieces were rehydrated in 10 mMDTT and 100 mM NH₄HCO₃, and incubated for 45 minutes at 56° C. Afterallowing the tubes to cool to room temperature, the liquid was removedand the same volume of a mixture of 55 mM iodoacetamide and 100 mMNH₄HCO₃ was immediately added. This was incubated for 30 minutes at roomtemperature in the dark. The liquid was removed, acetonitrile was addedto cover until the gel pieces turned a sticky white, at which time theacetonitrile was removed. The gel pieces were rehydrated in 100 mMNH₄HCO₃, and after 5 minutes, a volume of acetonitrile equal to twicethe volume of the gel pieces was added. This was incubated for 15minutes, the liquid removed, and the gel pieces dried in a Speed vac. Ifthe gel was stained with coomasie blue, and residual coomassie stillremained, the wash with 100 mM NH₄HCO₃/acetonitrile was repeated.

In-gel digestion. Gel pieces were completely dried down in a Speed Vac.The pieces were rehydrated in digestion buffer (50 mM NH₄HCO₃, 5 mMCaCl₂, 12.5 nanograms per microliter (ng/μl) trypsin) at 4° C. Enoughbuffer was added to cover the gel pieces, and more was added as needed.The gel pieces were incubated on ice for 45 minutes, and the supernatantremoved and replaced with 5-2 μl of same buffer without trypsin. Thiswas incubated at 37° C. overnight in an air incubator.

Extraction of peptides. A sufficient volume of 25 mM NH₄HCO₃ was addedto cover gel pieces, and incubated for 15 minutes (typically in a bathsonicator). The same volume of acetonitrile was added and incubated for15 minutes (in a bath sonicator if possible), and the supernatant wasrecovered. The extraction was repeated twice, using 5% formic acidinstead of NH₄HCO₃. A sufficient volume of 5% formic acid was added tocover gel pieces, and incubated for 15 minutes (typically in a bathsonicator). The same volume of acetonitrile was added and incubated for15 minutes (typically in a bath sonicator), and the supernatant wasrecovered. The extracts were pooled, and 10 mM DTT was added to a finalconcentration of 1 mM DTT. The sample was dried in a SpeedVac to a finalvolume of approximately 5 μl.

Desalting of peptides. The samples were desalted using a ZIPTIP pipettetips (C18, Millipore, Billerica, Mass.) as suggested by themanufacturer. Briefly, a sample was reconstituted in reconstitutionsolution (5:95 acetonitrile:H₂O, 0.1%-0.5% trifluoroacetic acid),centrifuged, and the pH checked to verify that it was less than 3. AZIPTIP was hydrated by aspirating 10 μl of solution 1 (50:50acetonitrile:H₂O, 0.1% trifluoroacetic acid) and discarding theaspirated aliquots. This was followed by aspirating 10 μl of solution 2(0.1% trifluoroacetic acid in deionized H₂O) and discarding theaspirated aliquots. The sample was loaded into the tip by aspirating 10μl of the sample slowly into the tip, expelling it into the sample tube,and repeating this 5 to 6 times. Ten microliters of solution 2 wasaspirated into the tip, the solution discarded by expelling, and thisprocess was repeated 5-7 times to wash. The peptides were eluted byaspirating 2.5 μl of ice cold solution 3 (60:40, acetonitrile:H₂O, 0.1%trofluoroacetic acid), expelling, and then re-aspirating the samealiquot in and out of the tip 3 times. After the solution has beenexpelled from the tip, the tube is capped and stored on ice.

Mass spectrometric peptide mapping. The peptides were suspended in 10 μlto 30 μl of 5% formic acid, and analyzed by MALDI-TOF MS (BrukerDaltonics Inc., Billerica, Mass.). The mass spectrum of the peptidefragments was determined as suggested by the manufacturer. Briefly, asample containing the peptides resulting from a tryptic digest weremixed with matrix cyano-4-hydroxycinnamic acid, transferred to a target,and allowed to dry. The dried sample was placed in the massspectrometer, irradiated, and the time of flight of each ion detectedand used to determine a peptide mass fingerprint for each proteinpresent in the composition. Known polypeptides were used to standardizethe machine.

Data analysis. The experimentally observed masses for the peptides ineach mass spectrum were compared to the expected masses of proteinsusing the Peptide Mass Fingerprint search method of the Mascot searchengine (Matrix Science Ltd., London, UK, and www.matrixscience.com, seePerkins et al., Electrophoresis 20, 3551-3567 (1999)). The searchparameters included: database, MSDB or NCBInr; taxonomy, bacteria(eubacteria) or Firmicutes (gram-positive bacteria); type of search,peptide mass fingerprint; enzyme, trypsin; fixed modifications,carbamidomethyl (C) or none; variable modifications, oxidation (M),carbamidomethyl (C), the combination, or none; mass values,monoisotopic; protein mass, unrestricted; peptide mass tolerance,between ±150 ppm and +430 ppm, or ±1 kDa; peptide charge state, Mr; maxmissed cleavages, 0 or 1; number of queries, 20.

Results

The result of this search was a mass fingerprint for each proteinpresent in the composition is shown in Tables 2, 3, 4, and 5.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference. The foregoing detaileddescription and examples have been given for clarity of understandingonly. No unnecessary limitations are to be understood therefrom. Theinvention is not limited to the exact details shown and described, forvariations obvious to one skilled in the art will be included within theinvention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

1-29. (canceled)
 30. An enriched first polypeptide having immunogenicactivity and a mass fingerprint at least 80% similar to a massfingerprint of a second polypeptide, wherein the second polypeptide hasa molecular weight of 88 kDa as determined by electrophoresis on asodium dodecyl sulfate-polyacrylamide gel and is expressed by aStaphylococcus aureus when incubated in media comprising an ironchelator and not expressed at a detectable level when grown in the mediawithout the iron chelator.
 31. An enriched polypeptide havingimmunogenic activity and a mass fingerprint that includes polypeptidefragments having one or more of m/z values 625.4, 717.3, 892.5, 942.5,944.5, 974.6, 984.5, 992.5, 1097.6, 1159.5, 1261.7, 1272.7, 1277.7,1289.7, 1315.7, 1322.7, 1394.7, 1417.8, 1421.8, 1426.8, 1508.8, 1513.9,1522.8, 1543.9, 1571.8, 1636.9, 1670.0, 1676.0, 1876.2, 2043.1, 2078.2,2285.5, and 2892.9.
 32. An enriched polypeptide having immunogenicactivity and an amino acid sequence having at least 80% similarity tothe amino acid sequence of SEQ ID NO:418.
 33. An enriched firstpolypeptide having immunogenic activity and a mass fingerprint at least80% similar to a mass fingerprint of a second polypeptide, wherein thesecond polypeptide has a molecular weight of 55 kDa as determined byelectrophoresis on a sodium dodecyl sulfate-polyacrylamide gel and isexpressed by a Staphylococcus aureus when incubated in media comprisingan iron chelator and not expressed at a detectable level when grown inthe media without the iron chelator.
 34. An enriched polypeptide havingimmunogenic activity and a mass fingerprint that includes polypeptidefragments having one or more of m/z values 783.6, 911.7, 937.6, 996.6,1025.6, 1063.6, 1185.6, 1277.6, 1324.7, 1346.7, 1381.8, 1394.8, 1400.7,1419.7, 1422.8, 1428.8, 1483.8, 1509.8, 1547.9, 1550.9, 1559.9, 1788.1,1930.1, 1946.0, 2100.4, 2239.3, 2493.5, 2900.6, and 2916.6.
 35. Anenriched polypeptide having immunogenic activity and an amino acidsequence having at least 80% similarity to the amino acid sequence ofSEQ ID NO:419.
 36. An enriched first polypeptide having immunogenicactivity and a mass fingerprint at least 80% similar to a massfingerprint of a second polypeptide, wherein the second polypeptide hasa molecular weight of 38 kDa as determined by electrophoresis on asodium dodecyl sulfate-polyacrylamide gel and is expressed by aStaphylococcus aureus when incubated in media comprising an ironchelator and not expressed at a detectable level when grown in the mediawithout the iron chelator.
 37. An enriched polypeptide havingimmunogenic activity and a mass fingerprint that includes polypeptidefragments having one or more of m/z values 993.6, 996.7, 1237.7, 1272.7,1502.0, 1507.9, 1523.9, 1559.9, 1716.0, 1737.0, 1844.1, 1929.1, 1998.2,2234.4, and 3143.8.
 38. An enriched polypeptide having immunogenicactivity and an amino acid sequence having at least 80% similarity tothe amino acid sequence of SEQ ID NO:420.
 39. An enriched firstpolypeptide having immunogenic activity and a mass fingerprint at least80% similar to a mass fingerprint of a second polypeptide, wherein thesecond polypeptide has a molecular weight of 37 kDa as determined byelectrophoresis on a sodium dodecyl sulfate-polyacrylamide gel and isexpressed by a Staphylococcus aureus when incubated in media comprisingan iron chelator and not expressed at a detectable level when grown inthe media without the iron chelator.
 40. An enriched polypeptide havingimmunogenic activity and a mass fingerprint that includes polypeptidefragments having one or more of m/z values 699.5, 729.4, 792.5, 852.4,987.5, 1008.5, 1020.5, 1074.5, 1083.6, 1169.5, 1182.5, 1184.5, 1223.5,1278.6, 1497.6, 1502.7, 1558.8, 1605.8, 1623.8, 1712.8, 1800.9, 1957.0,2252.0, and 3383.9.
 41. An enriched polypeptide having immunogenicactivity and an amino acid sequence having at least 80% similarity tothe amino acid sequence of SEQ ID NO:421.
 42. An enriched firstpolypeptide having immunogenic activity and a mass fingerprint at least80% similar to a mass fingerprint of a second polypeptide, wherein thesecond polypeptide has a molecular weight of 36 kDa as determined byelectrophoresis on a sodium dodecyl sulfate-polyacrylamide gel and isexpressed by a Staphylococcus aureus when incubated in media comprisingan iron chelator and not expressed at a detectable level when grown inthe media without the iron chelator.
 43. An enriched polypeptide havingimmunogenic activity and a mass fingerprint that includes polypeptidefragments having one or more of m/z values 646.4, 725.5, 1068.4, 1185.5,1327.6, 1343.6, 2080.9, 2438.1, and 2789.4.
 44. An enriched polypeptidehaving immunogenic activity and an amino acid sequence having at least80% similarity to the amino acid sequence of SEQ ID NO:422.
 45. Anenriched first polypeptide having immunogenic activity and a massfingerprint at least 80% similar to a mass fingerprint of a secondpolypeptide, wherein the second polypeptide has a molecular weight of 35kDa as determined by electrophoresis on a sodium dodecylsulfate-polyacrylamide gel and is expressed by a Staphylococcus aureuswhen incubated in media comprising an iron chelator and not expressed ata detectable level when grown in the media without the iron chelator.46. An enriched polypeptide having immunogenic activity and a massfingerprint that includes polypeptide fragments having one or more ofm/z values 760.5, 1012.6, 1107.6, 1204.7, 1238.6, 1244.6, 1259.7,1281.7, 1516.8, 1683.9, 1877.1, 1884.0, 2227.1, and 2781.4.
 47. Anenriched polypeptide having immunogenic activity and an amino acidsequence having at least 80% similarity to the amino acid sequence ofSEQ ID NO:423.
 48. An enriched first polypeptide having immunogenicactivity and a mass fingerprint at least 80% similar to a massfingerprint of a second polypeptide, wherein the second polypeptide hasa molecular weight of 33 kDa as determined by electrophoresis on asodium dodecyl sulfate-polyacrylamide gel and is expressed by aStaphylococcus aureus when incubated in media comprising an ironchelator and not expressed at a detectable level when grown in the mediawithout the iron chelator.
 49. An enriched polypeptide havingimmunogenic activity and a mass fingerprint that includes polypeptidefragments having one or more of m/z values 834.5, 864.5, 946.5, 962.5,976.5, 1054.5, 1202.5, 1268.6, 1443.6, 1450.7, 1454.7, 1571.7, 1593.7,1818.9, 1836.9, 1911.9, and 2582.3.
 50. An enriched polypeptide havingimmunogenic activity and an amino acid sequence having at least 80%similarity to the amino acid sequence of SEQ ID NO:424.